Perform Downstream Analysis

This notebook is for performing downstream analysis of CYCLOPS. It includes running cosinor regression and assessing differential rhythmicity (two ways for both), calling the enrichR API via python, using fgsea in R, running PSEA, outputing files for making KEGG map diagrams, etc.

Load Packages

This notebook makes use of python via anaconda. Please make sure you install anaconda to proceed:

conda_list()

Select your conda environment of choice, in my case, item 2 from list above:

conda_list()[[2]][2] %>% 
  use_condaenv(required = TRUE)

Warning: Previous request to `use_python("/usr/bin/python3", required = TRUE)` will be ignored. It is superseded by request to `use_python("/Users/henryhollis/opt/anaconda3/bin/python")

print(paste("Using cond env:",conda_list()[[1]][2] ))

[1] "Using cond env: base"

Setup

Select the CYCLOPS ordering path and path to data file and path to DEseq2 differential expression results:

#path to cyclops ordering
path_to_cyclops_ordering = "/Users/henryhollis/Box Sync/Henry_stuff/AD_project/human_data/Cyclops_folders/training_output/scROSMAP/cogdx_controls/wAD/ExcitatoryNeurons/TMMs_w_batch/Exc3and5_FiltByEdgeRDefault_fixedipBulkChenZhang_condAndBatchCovs_04ContrVar4EGdefault_noTransferFit_Feb5Redo/"

#path to normalized counts
path_to_tmm_file = "/Users/henryhollis/Box Sync/Henry_stuff/AD_project/human_data/Cyclops_folders/tmms/scROSMAP/cogdx_controls/EdgeR_filt_normed/Microglia_4_5_6_7_8_10_11_13_15_16_FiltByExprDefault_TMM.csv"

# optional path to file containing differential expression results
deseq_de_filename = "/Users/henryhollis/Box Sync/Henry_stuff/AD_project/scROSMAP/simple_differential_expr/Mglia_4_5_6_7_8_10_11_13_15_16/ Microglia_4_5_6_7_8_10_11_13_15_16_filtByExpr_combatSeq_edgeR_DE_exactTest.csv"

path_to_rosmap_clin = "~/Box Sync/Henry_stuff/AD_project/scROSMAP/Meta_data/cleaned_rosmap_meta_cogdxConds.csv"
metabolon_filename = "~/Box Sync/Henry_stuff/AD_project/scROSMAP/Metabolon/ROSMAP_Metabolon_HD4_Brain514_assay_data.csv"
metabolon_datakey = "~/Box Sync/Henry_stuff/AD_project/scROSMAP/Metabolon/ROSMAP_Metabolon_HD4_Data_Dictionary.csv"

#BHQ corrected p cutoff to be considered cycling in analysis:
isCyclingBHQCutoff = 0.1
percentile = 0.025
useBatch = T

Cosinor Regression and Differential Rhythmicity

Run cosinor regression and differential rhythmicity analysis with custom script and with compareRhythms library. I make use of Ensembl and Entrez IDs so I need the following translation dictionaries:

# read in translation dict between gene symbols and other alias
Ensembl_dict = readxl::read_xlsx("scROSMAP_ENSEMBL_dict.xlsx")

#run custom cycling and differential rhythmicity analysis
print("***Running cosinor and differential rhythms analysis***")

[1] "***Running cosinor and differential rhythms analysis***"

run_cycling_and_dr_analysis(path_to_cyclops_ordering, path_to_tmm_file, isCyclingSigCutoff = isCyclingBHQCutoff, percentile = percentile, useBatch = useBatch)

Running is_cycling() on cond_subset: cond_0
NOTE: Using batches in regression.

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Running is_cycling() on cond_subset: cond_1
NOTE: Using batches in regression.

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Running is_cycling_method2() (analogue of compareRhythms) on all subjects
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Running Mesor_differences()
NOTE: Using batches in regression.

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Running diff_rhyth_AD_severity()
NOTE: Using batches in regression.

Warning: The `x` argument of `as_tibble.matrix()` must have unique column names if `.name_repair` is omitted as of tibble 2.0.0.
Using compatibility `.name_repair`.

Running diff_rhyth() on seedlist of size: 282
NOTE: Using batches in regression.

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Running diff_rhyth() on seedlist of size: 110
NOTE: Using batches in regression.

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Running diff_rhyth() on seedlist of size: 3130
NOTE: Using batches in regression.

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NOTE: Using batches in regression.

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NOTE: Using batches in regression.

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Plotting

With custom regression, I have to remake clock face plots, genes plots, etc.

setwd(path_to_cyclops_ordering)

Warning: The working directory was changed to /Users/henryhollis/Box Sync/Henry_stuff/AD_project/human_data/Cyclops_folders/training_output/scROSMAP/cogdx_controls/wAD/ExcitatoryNeurons/TMMs_w_batch/Exc3and5_FiltByEdgeRDefault_fixedipBulkChenZhang_condAndBatchCovs_04ContrVar4EGdefault_noTransferFit_Feb5Redo inside a notebook chunk. The working directory will be reset when the chunk is finished running. Use the knitr root.dir option in the setup chunk to change the working directory for notebook chunks.

if (!(dir.exists("downstream_output/plots"))){
  dir.create("downstream_output/plots")
}

#for cycling_in_CTL.csv
plot_clock_face(paste0(path_to_cyclops_ordering, "/downstream_output/plots/CTL_clock_face_plot.png"), "downstream_output/cosinor_results_CTL.csv", mouse_data = mouse_data, BHQ_cutoff = isCyclingBHQCutoff, amp_ratio_cutoff = 0, force_align = 5.907, force_flipped = T)

[1] "Shifting phases using `force align`"
[1] "flipping acrophases."
NULL

setwd(path_to_cyclops_ordering)

#for cycling_in_AD.csv
plot_clock_face(paste0(path_to_cyclops_ordering, "/downstream_output/plots/AD_clock_face_plot.png"),"downstream_output/cosinor_results_AD.csv",mouse_data = mouse_data, BHQ_cutoff = isCyclingBHQCutoff, amp_ratio_cutoff = 0, force_align = 5.907, force_flipped = T)

[1] "Shifting phases using `force align`"
[1] "flipping acrophases."
NULL

#plot cyclops histogram
plot_subject_histogram(path_to_cyclops_ordering, "cond_0")

null device 
          1 

plot_subject_histogram(path_to_cyclops_ordering, "cond_1")

null device 
          1 

#Gene Tracings
plot_core_clock_genes(path_to_tmm_file, path_to_cyclops_ordering, useBatch = useBatch, percentile = percentile, split_cond_plots = T)

[1] "NOTE: Using batches in plotting"

Plot Other Genes

plot_me = c("RPL18","RPS7","RPL22","RPS18","RPL8","RPL10A","RPS13","RPL14","RPS15A","RPS12","RPL36AL","RPL34","RPL30","RPL9","RPL21","RPS28","RPSA","RPL26","RPL27A","FAU","RPS3A","RPL7A","RPL17","RPS27A","RPS16","RPL15","RPLP1","RPL7","RPL10","RPL23A","RPL18A","RPL3","RPL12","RPL39","RPL37A","RPL37","RPS10","RPL13","RPL35A","RPS23","RPL13A","RPL38","RPS29","RPL24","RPL41","RPL35","RPS26","RPS2","RPS9","RPL32","RPL6","RPS21","RPS24","RPS25","RPL36","RPL19","RPS27","RPL29","RPS15","RPS3","RPS5","RPS8","RPL27","RSL24D1","RPS6","RPL11")

tmm = read_csv(path_to_tmm_file, show_col_types = FALSE)
  cyc_pred_file = list.files(path = paste0(path_to_cyclops_ordering, "/Fits/"), pattern = '*Fit_Output_*')
cyc_pred = read_csv(paste(path_to_cyclops_ordering, "Fits", cyc_pred_file[1], sep = '/'), show_col_types = FALSE)
plot_gene_trace(cyc_pred, tmm, plot_me , percentile = percentile, useBatch = useBatch)

[1] "NOTE: Using batches in plotting"

EnrichR

Call EnrichR API with python, supplying various lists from cycling and differential rhythm analysis.

path = getwd()
setwd(paste0(path_to_cyclops_ordering, "downstream_output"))

Warning: The working directory was changed to /Users/henryhollis/Box Sync/Henry_stuff/AD_project/human_data/Cyclops_folders/training_output/scROSMAP/cogdx_controls/wAD/ExcitatoryNeurons/TMMs_w_batch/Exc3and5_FiltByEdgeRDefault_fixedipBulkChenZhang_condAndBatchCovs_04ContrVar4EGdefault_noTransferFit_Feb5Redo/downstream_output inside a notebook chunk. The working directory will be reset when the chunk is finished running. Use the knitr root.dir option in the setup chunk to change the working directory for notebook chunks.

path_no_space = gsub(" ", "\\ ", path, fixed = TRUE)
#create list of filenames from the enrichR_files subfolder
gene_lists = list.files("./enrichR_files/", pattern = "^(CTL_|AD_|diff_mesor)")

#run system command to call python script that uses enrichR API
sapply(gene_lists, function(x){ 
  system(paste0("python3 ", path_no_space, "/Python_EnrichR_for_AnalysisPipeline.py -g \"enrichR_files/" ,x, "\" -b \"enrichR_files/EnrichR_background.csv\""))})

/Users/henryhollis/Box Sync/Henry_stuff/AD_project/scROSMAP/Rscripts/automatic_downstream_analysis/Python_EnrichR_for_AnalysisPipeline.py:177: RuntimeWarning: More than 20 figures have been opened. Figures created through the pyplot interface (`matplotlib.pyplot.figure`) are retained until explicitly closed and may consume too much memory. (To control this warning, see the rcParam `figure.max_open_warning`).
  fig, axes = plt.subplots(2, 2, figsize=(15, 10))

{'lib': ['KEGG_2021_Human', 'GO_Biological_Process_2023', 'MSigDB_Hallmark_2020', 'WikiPathway_2021_Human', 'TRRUST_Transcription_Factors_2019', 'GO_Cellular_Component_2023', 'TRANSFAC_and_JASPAR_PWMs', 'Transcription_Factor_PPIs', 'TargetScan_microRNA_2017', 'miRTarBase_2017'], 'genes_list_file': 'enrichR_files/AD_CTL_mthd2_cyclers_AR25BHQ1.csv', 'background_list_file': 'enrichR_files/EnrichR_background.csv'}
Please select file containing list of genes (Symbols)
Path to genes list: enrichR_files/AD_CTL_mthd2_cyclers_AR25BHQ1.csv

Please select file containing list of BACKGROUND genes (Symbols)
Path to Background list: enrichR_files/EnrichR_background.csv
Background DataFrame (nrow: 301):
           Ensembl Gene_Symbols
0  ENSG00000142920        AZIN2
1  ENSG00000132639       SNAP25
2  ENSG00000168329       CX3CR1
3  ENSG00000166833         NAV2
4  ENSG00000213316        LTC4S
Background DataFrame (nrow: 6966):
           Ensembl Gene_Symbols
0  ENSG00000078808         SDF4
1  ENSG00000160087       UBE2J2
2  ENSG00000131584        ACAP3
3  ENSG00000127054       INTS11
4  ENSG00000175756     AURKAIP1
Directory "EnrichR_results/EnrichR_ADCTLmthd2cyclersAR25BHQ1_BCKGRND-EnrichRbackground" created.
Saved KEGG_2021_Human results to file
Saved GO_Biological_Process_2023 results to file
Saved MSigDB_Hallmark_2020 results to file
Saved WikiPathway_2021_Human results to file
Saved TRRUST_Transcription_Factors_2019 results to file
Saved GO_Cellular_Component_2023 results to file
Saved TRANSFAC_and_JASPAR_PWMs results to file
Saved Transcription_Factor_PPIs results to file
Saved TargetScan_microRNA_2017 results to file
Saved miRTarBase_2017 results to file

/Users/henryhollis/Box Sync/Henry_stuff/AD_project/scROSMAP/Rscripts/automatic_downstream_analysis/Python_EnrichR_for_AnalysisPipeline.py:177: RuntimeWarning: More than 20 figures have been opened. Figures created through the pyplot interface (`matplotlib.pyplot.figure`) are retained until explicitly closed and may consume too much memory. (To control this warning, see the rcParam `figure.max_open_warning`).
  fig, axes = plt.subplots(2, 2, figsize=(15, 10))

{'lib': ['KEGG_2021_Human', 'GO_Biological_Process_2023', 'MSigDB_Hallmark_2020', 'WikiPathway_2021_Human', 'TRRUST_Transcription_Factors_2019', 'GO_Cellular_Component_2023', 'TRANSFAC_and_JASPAR_PWMs', 'Transcription_Factor_PPIs', 'TargetScan_microRNA_2017', 'miRTarBase_2017'], 'genes_list_file': 'enrichR_files/AD_cyclers_AR25BHQ1.csv', 'background_list_file': 'enrichR_files/EnrichR_background.csv'}
Please select file containing list of genes (Symbols)
Path to genes list: enrichR_files/AD_cyclers_AR25BHQ1.csv

Please select file containing list of BACKGROUND genes (Symbols)
Path to Background list: enrichR_files/EnrichR_background.csv
Background DataFrame (nrow: 197):
           Ensembl Gene_Symbols
0  ENSG00000132639       SNAP25
1  ENSG00000142920        AZIN2
2  ENSG00000017427         IGF1
3  ENSG00000166833         NAV2
4  ENSG00000213316        LTC4S
Background DataFrame (nrow: 6966):
           Ensembl Gene_Symbols
0  ENSG00000078808         SDF4
1  ENSG00000160087       UBE2J2
2  ENSG00000131584        ACAP3
3  ENSG00000127054       INTS11
4  ENSG00000175756     AURKAIP1
Directory "EnrichR_results/EnrichR_ADcyclersAR25BHQ1_BCKGRND-EnrichRbackground" created.
Saved KEGG_2021_Human results to file
Saved GO_Biological_Process_2023 results to file
Saved MSigDB_Hallmark_2020 results to file
Saved WikiPathway_2021_Human results to file
Saved TRRUST_Transcription_Factors_2019 results to file
Saved GO_Cellular_Component_2023 results to file
Saved TRANSFAC_and_JASPAR_PWMs results to file
Saved Transcription_Factor_PPIs results to file
Saved TargetScan_microRNA_2017 results to file
Saved miRTarBase_2017 results to file

/Users/henryhollis/Box Sync/Henry_stuff/AD_project/scROSMAP/Rscripts/automatic_downstream_analysis/Python_EnrichR_for_AnalysisPipeline.py:177: RuntimeWarning: More than 20 figures have been opened. Figures created through the pyplot interface (`matplotlib.pyplot.figure`) are retained until explicitly closed and may consume too much memory. (To control this warning, see the rcParam `figure.max_open_warning`).
  fig, axes = plt.subplots(2, 2, figsize=(15, 10))

{'lib': ['KEGG_2021_Human', 'GO_Biological_Process_2023', 'MSigDB_Hallmark_2020', 'WikiPathway_2021_Human', 'TRRUST_Transcription_Factors_2019', 'GO_Cellular_Component_2023', 'TRANSFAC_and_JASPAR_PWMs', 'Transcription_Factor_PPIs', 'TargetScan_microRNA_2017', 'miRTarBase_2017'], 'genes_list_file': 'enrichR_files/AD_cyclers_AR33BHQ1.csv', 'background_list_file': 'enrichR_files/EnrichR_background.csv'}
Please select file containing list of genes (Symbols)
Path to genes list: enrichR_files/AD_cyclers_AR33BHQ1.csv

Please select file containing list of BACKGROUND genes (Symbols)
Path to Background list: enrichR_files/EnrichR_background.csv
Background DataFrame (nrow: 80):
           Ensembl Gene_Symbols
0  ENSG00000142920        AZIN2
1  ENSG00000017427         IGF1
2  ENSG00000166833         NAV2
3  ENSG00000213316        LTC4S
4  ENSG00000019485       PRDM11
Background DataFrame (nrow: 6966):
           Ensembl Gene_Symbols
0  ENSG00000078808         SDF4
1  ENSG00000160087       UBE2J2
2  ENSG00000131584        ACAP3
3  ENSG00000127054       INTS11
4  ENSG00000175756     AURKAIP1
Directory "EnrichR_results/EnrichR_ADcyclersAR33BHQ1_BCKGRND-EnrichRbackground" created.
Saved KEGG_2021_Human results to file
Saved GO_Biological_Process_2023 results to file
Saved MSigDB_Hallmark_2020 results to file
Saved WikiPathway_2021_Human results to file
Saved TRRUST_Transcription_Factors_2019 results to file
Saved GO_Cellular_Component_2023 results to file
Saved TRANSFAC_and_JASPAR_PWMs results to file
Saved Transcription_Factor_PPIs results to file
Saved TargetScan_microRNA_2017 results to file
Saved miRTarBase_2017 results to file

/Users/henryhollis/Box Sync/Henry_stuff/AD_project/scROSMAP/Rscripts/automatic_downstream_analysis/Python_EnrichR_for_AnalysisPipeline.py:177: RuntimeWarning: More than 20 figures have been opened. Figures created through the pyplot interface (`matplotlib.pyplot.figure`) are retained until explicitly closed and may consume too much memory. (To control this warning, see the rcParam `figure.max_open_warning`).
  fig, axes = plt.subplots(2, 2, figsize=(15, 10))

{'lib': ['KEGG_2021_Human', 'GO_Biological_Process_2023', 'MSigDB_Hallmark_2020', 'WikiPathway_2021_Human', 'TRRUST_Transcription_Factors_2019', 'GO_Cellular_Component_2023', 'TRANSFAC_and_JASPAR_PWMs', 'Transcription_Factor_PPIs', 'TargetScan_microRNA_2017', 'miRTarBase_2017'], 'genes_list_file': 'enrichR_files/CTL_cyclers_AR25BHQ1.csv', 'background_list_file': 'enrichR_files/EnrichR_background.csv'}
Please select file containing list of genes (Symbols)
Path to genes list: enrichR_files/CTL_cyclers_AR25BHQ1.csv

Please select file containing list of BACKGROUND genes (Symbols)
Path to Background list: enrichR_files/EnrichR_background.csv
Background DataFrame (nrow: 204):
           Ensembl Gene_Symbols
0  ENSG00000168329       CX3CR1
1  ENSG00000142920        AZIN2
2  ENSG00000196683        TOMM7
3  ENSG00000070087         PFN2
4  ENSG00000166833         NAV2
Background DataFrame (nrow: 6966):
           Ensembl Gene_Symbols
0  ENSG00000078808         SDF4
1  ENSG00000160087       UBE2J2
2  ENSG00000131584        ACAP3
3  ENSG00000127054       INTS11
4  ENSG00000175756     AURKAIP1
Directory "EnrichR_results/EnrichR_CTLcyclersAR25BHQ1_BCKGRND-EnrichRbackground" created.
Saved KEGG_2021_Human results to file
Saved GO_Biological_Process_2023 results to file
Saved MSigDB_Hallmark_2020 results to file
Saved WikiPathway_2021_Human results to file
Saved TRRUST_Transcription_Factors_2019 results to file
Saved GO_Cellular_Component_2023 results to file
Saved TRANSFAC_and_JASPAR_PWMs results to file
Saved Transcription_Factor_PPIs results to file
Saved TargetScan_microRNA_2017 results to file
Saved miRTarBase_2017 results to file

/Users/henryhollis/Box Sync/Henry_stuff/AD_project/scROSMAP/Rscripts/automatic_downstream_analysis/Python_EnrichR_for_AnalysisPipeline.py:177: RuntimeWarning: More than 20 figures have been opened. Figures created through the pyplot interface (`matplotlib.pyplot.figure`) are retained until explicitly closed and may consume too much memory. (To control this warning, see the rcParam `figure.max_open_warning`).
  fig, axes = plt.subplots(2, 2, figsize=(15, 10))

{'lib': ['KEGG_2021_Human', 'GO_Biological_Process_2023', 'MSigDB_Hallmark_2020', 'WikiPathway_2021_Human', 'TRRUST_Transcription_Factors_2019', 'GO_Cellular_Component_2023', 'TRANSFAC_and_JASPAR_PWMs', 'Transcription_Factor_PPIs', 'TargetScan_microRNA_2017', 'miRTarBase_2017'], 'genes_list_file': 'enrichR_files/CTL_cyclers_AR33BHQ1.csv', 'background_list_file': 'enrichR_files/EnrichR_background.csv'}
Please select file containing list of genes (Symbols)
Path to genes list: enrichR_files/CTL_cyclers_AR33BHQ1.csv

Please select file containing list of BACKGROUND genes (Symbols)
Path to Background list: enrichR_files/EnrichR_background.csv
Background DataFrame (nrow: 74):
           Ensembl Gene_Symbols
0  ENSG00000168329       CX3CR1
1  ENSG00000142920        AZIN2
2  ENSG00000166833         NAV2
3  ENSG00000019485       PRDM11
4  ENSG00000213316        LTC4S
Background DataFrame (nrow: 6966):
           Ensembl Gene_Symbols
0  ENSG00000078808         SDF4
1  ENSG00000160087       UBE2J2
2  ENSG00000131584        ACAP3
3  ENSG00000127054       INTS11
4  ENSG00000175756     AURKAIP1
Directory "EnrichR_results/EnrichR_CTLcyclersAR33BHQ1_BCKGRND-EnrichRbackground" created.
Saved KEGG_2021_Human results to file
Saved GO_Biological_Process_2023 results to file
Saved MSigDB_Hallmark_2020 results to file
Saved WikiPathway_2021_Human results to file
Saved TRRUST_Transcription_Factors_2019 results to file
Saved GO_Cellular_Component_2023 results to file
Saved TRANSFAC_and_JASPAR_PWMs results to file
Saved Transcription_Factor_PPIs results to file
Saved TargetScan_microRNA_2017 results to file
Saved miRTarBase_2017 results to file

/Users/henryhollis/Box Sync/Henry_stuff/AD_project/scROSMAP/Rscripts/automatic_downstream_analysis/Python_EnrichR_for_AnalysisPipeline.py:177: RuntimeWarning: More than 20 figures have been opened. Figures created through the pyplot interface (`matplotlib.pyplot.figure`) are retained until explicitly closed and may consume too much memory. (To control this warning, see the rcParam `figure.max_open_warning`).
  fig, axes = plt.subplots(2, 2, figsize=(15, 10))

{'lib': ['KEGG_2021_Human', 'GO_Biological_Process_2023', 'MSigDB_Hallmark_2020', 'WikiPathway_2021_Human', 'TRRUST_Transcription_Factors_2019', 'GO_Cellular_Component_2023', 'TRANSFAC_and_JASPAR_PWMs', 'Transcription_Factor_PPIs', 'TargetScan_microRNA_2017', 'miRTarBase_2017'], 'genes_list_file': 'enrichR_files/diff_mesor_all_genes_BHQ05.csv', 'background_list_file': 'enrichR_files/EnrichR_background.csv'}
Please select file containing list of genes (Symbols)
Path to genes list: enrichR_files/diff_mesor_all_genes_BHQ05.csv

Please select file containing list of BACKGROUND genes (Symbols)
Path to Background list: enrichR_files/EnrichR_background.csv
Background DataFrame (nrow: 34):
           Ensembl Gene_Symbols
0  ENSG00000188641         DPYD
1  ENSG00000152689      RASGRP3
2  ENSG00000121964        GTDC1
3  ENSG00000116117       PARD3B
4  ENSG00000131389       SLC6A6
Background DataFrame (nrow: 6966):
           Ensembl Gene_Symbols
0  ENSG00000078808         SDF4
1  ENSG00000160087       UBE2J2
2  ENSG00000131584        ACAP3
3  ENSG00000127054       INTS11
4  ENSG00000175756     AURKAIP1
Directory "EnrichR_results/EnrichR_diffmesorallgenesBHQ05_BCKGRND-EnrichRbackground" created.
Saved KEGG_2021_Human results to file
Saved GO_Biological_Process_2023 results to file
Saved MSigDB_Hallmark_2020 results to file
Saved WikiPathway_2021_Human results to file
Saved TRRUST_Transcription_Factors_2019 results to file
Saved GO_Cellular_Component_2023 results to file
Saved TRANSFAC_and_JASPAR_PWMs results to file
Saved Transcription_Factor_PPIs results to file
Saved TargetScan_microRNA_2017 results to file
Saved miRTarBase_2017 results to file
AD_CTL_mthd2_cyclers_AR25BHQ1.csv           AD_cyclers_AR25BHQ1.csv           AD_cyclers_AR33BHQ1.csv          CTL_cyclers_AR25BHQ1.csv 
                                0                                 0                                 0                                 0 
         CTL_cyclers_AR33BHQ1.csv    diff_mesor_all_genes_BHQ05.csv 
                                0                                 0 

isCyclingBHQCutoff_str = str_extract(as.character(isCyclingBHQCutoff), "(?<=\\.)\\d+")

setwd("enrichR_files/")
all_DR_gene_lists = list.files( pattern = "^(DR_)")
method2_files = grepl("mthd2", all_DR_gene_lists)
DR_gene_lists_method1 = all_DR_gene_lists[!method2_files]
DR_backgrounds_method1 = paste0("diff_rhythms_CyclingBHQ", isCyclingBHQCutoff_str, "AmpRatio", str_replace(str_extract(DR_gene_lists_method1, pattern = "AR\\d+"), "AR", ""), ".csv")
DR_gene_lists_method2 = all_DR_gene_lists[method2_files]
DR_backgrounds_method2 = paste0("diff_rhythms_method2_CyclingBHQ", isCyclingBHQCutoff_str, "AmpRatio", str_replace(str_extract(DR_gene_lists_method2, pattern = "AR\\d+"), "AR", ""), ".csv")
DR_gene_lists = c(DR_gene_lists_method1, DR_gene_lists_method2)
DR_backgrounds = c(DR_backgrounds_method1, DR_backgrounds_method2)

# repeat with differential gene lists
setwd(paste0(path_to_cyclops_ordering, "downstream_output"))
mapply(function(x, y){ 
  system(paste0("python3 ", path_no_space, "/Python_EnrichR_for_AnalysisPipeline.py -g \"enrichR_files/" ,x, "\" -b \"",y , "\""))},
  DR_gene_lists, 
  DR_backgrounds)

/Users/henryhollis/Box Sync/Henry_stuff/AD_project/scROSMAP/Rscripts/automatic_downstream_analysis/Python_EnrichR_for_AnalysisPipeline.py:177: RuntimeWarning: More than 20 figures have been opened. Figures created through the pyplot interface (`matplotlib.pyplot.figure`) are retained until explicitly closed and may consume too much memory. (To control this warning, see the rcParam `figure.max_open_warning`).
  fig, axes = plt.subplots(2, 2, figsize=(15, 10))

{'lib': ['KEGG_2021_Human', 'GO_Biological_Process_2023', 'MSigDB_Hallmark_2020', 'WikiPathway_2021_Human', 'TRRUST_Transcription_Factors_2019', 'GO_Cellular_Component_2023', 'TRANSFAC_and_JASPAR_PWMs', 'Transcription_Factor_PPIs', 'TargetScan_microRNA_2017', 'miRTarBase_2017'], 'genes_list_file': 'enrichR_files/DR_cyclers_CyclingBHQ1AR1_DRBHQ2.csv', 'background_list_file': 'diff_rhythms_CyclingBHQ1AmpRatio1.csv'}
Please select file containing list of genes (Symbols)
Path to genes list: enrichR_files/DR_cyclers_CyclingBHQ1AR1_DRBHQ2.csv

Please select file containing list of BACKGROUND genes (Symbols)
Path to Background list: diff_rhythms_CyclingBHQ1AmpRatio1.csv
Background DataFrame (nrow: 12):
           Ensembl Gene_Symbols  ...      Bonf  Log_AD_CTL_ampRatio
0  ENSG00000136715       SAP130  ...  0.409230             0.238614
1  ENSG00000127990         SGCE  ...  1.000000             0.866906
2  ENSG00000164985        PSIP1  ...  0.958730             2.777088
3  ENSG00000119414        PPP6C  ...  1.000000             1.002309
4  ENSG00000148606       POLR3A  ...  0.498371             0.654147

[5 rows x 10 columns]
Background DataFrame (nrow: 3130):
           Ensembl Gene_Symbols         p  ...       BHQ  Bonf  Log_AD_CTL_ampRatio
0  ENSG00000131584        ACAP3  0.669440  ...  0.861040   1.0            -0.772190
1  ENSG00000175756     AURKAIP1  0.526738  ...  0.796164   1.0             0.185463
2  ENSG00000142609       CFAP74  0.238942  ...  0.629488   1.0            -0.349632
3  ENSG00000187730        GABRD  0.490882  ...  0.786366   1.0             0.532005
4  ENSG00000158109       TPRG1L  0.834309  ...  0.933889   1.0            -0.279667

[5 rows x 10 columns]
Directory "EnrichR_results/EnrichR_DRcyclersCyclingBHQ1AR1DRBHQ2_BCKGRND-diffrhythmsCyclingBHQ1AmpRatio1" created.
Saved KEGG_2021_Human results to file
Saved GO_Biological_Process_2023 results to file
Saved MSigDB_Hallmark_2020 results to file
Saved WikiPathway_2021_Human results to file
Saved TRRUST_Transcription_Factors_2019 results to file
Saved GO_Cellular_Component_2023 results to file
Saved TRANSFAC_and_JASPAR_PWMs results to file
Saved Transcription_Factor_PPIs results to file
Saved TargetScan_microRNA_2017 results to file
Saved miRTarBase_2017 results to file
{'lib': ['KEGG_2021_Human', 'GO_Biological_Process_2023', 'MSigDB_Hallmark_2020', 'WikiPathway_2021_Human', 'TRRUST_Transcription_Factors_2019', 'GO_Cellular_Component_2023', 'TRANSFAC_and_JASPAR_PWMs', 'Transcription_Factor_PPIs', 'TargetScan_microRNA_2017', 'miRTarBase_2017'], 'genes_list_file': 'enrichR_files/DR_cyclers_CyclingBHQ1AR25_DRBHQ2.csv', 'background_list_file': 'diff_rhythms_CyclingBHQ1AmpRatio25.csv'}
Please select file containing list of genes (Symbols)
Path to genes list: enrichR_files/DR_cyclers_CyclingBHQ1AR25_DRBHQ2.csv

Please select file containing list of BACKGROUND genes (Symbols)
Path to Background list: diff_rhythms_CyclingBHQ1AmpRatio25.csv
Background DataFrame (nrow: 7):
           Ensembl Gene_Symbols  ...      Bonf  Log_AD_CTL_ampRatio
0  ENSG00000148219        ASTN2  ...  0.127360             1.276032
1  ENSG00000140470     ADAMTS17  ...  0.066427             2.177640
2  ENSG00000158258       CLSTN2  ...  0.517345            -0.586008
3  ENSG00000095261        PSMD5  ...  0.368817             1.203439
4  ENSG00000134419       RPS15A  ...  0.431026            -1.276418

[5 rows x 10 columns]
Background DataFrame (nrow: 282):
           Ensembl Gene_Symbols         p  ...       BHQ  Bonf  Log_AD_CTL_ampRatio
0  ENSG00000097021        ACOT7  0.009033  ...  0.282688   1.0             1.408467
1  ENSG00000171680      PLEKHG5  0.086548  ...  0.488133   1.0             0.943269
2  ENSG00000049246         PER3  0.252187  ...  0.700387   1.0             0.326548
3  ENSG00000028137     TNFRSF1B  0.357533  ...  0.741148   1.0             0.499864
4  ENSG00000173436      MICOS10  0.628646  ...  0.864771   1.0             0.217262

[5 rows x 10 columns]
Directory "EnrichR_results/EnrichR_DRcyclersCyclingBHQ1AR25DRBHQ2_BCKGRND-diffrhythmsCyclingBHQ1AmpRatio25" created.
Saved KEGG_2021_Human results to file
Saved GO_Biological_Process_2023 results to file
{'lib': ['KEGG_2021_Human', 'GO_Biological_Process_2023', 'MSigDB_Hallmark_2020', 'WikiPathway_2021_Human', 'TRRUST_Transcription_Factors_2019', 'GO_Cellular_Component_2023', 'TRANSFAC_and_JASPAR_PWMs', 'Transcription_Factor_PPIs', 'TargetScan_microRNA_2017', 'miRTarBase_2017'], 'genes_list_file': 'enrichR_files/DR_cyclers_CyclingBHQ1AR33_DRBHQ2.csv', 'background_list_file': 'diff_rhythms_CyclingBHQ1AmpRatio33.csv'}
Please select file containing list of genes (Symbols)
Path to genes list: enrichR_files/DR_cyclers_CyclingBHQ1AR33_DRBHQ2.csv

Please select file containing list of BACKGROUND genes (Symbols)
Path to Background list: diff_rhythms_CyclingBHQ1AmpRatio33.csv
Background DataFrame (nrow: 2):
           Ensembl Gene_Symbols  ...      Bonf  Log_AD_CTL_ampRatio
0  ENSG00000140470     ADAMTS17  ...  0.025911             2.177640
1  ENSG00000269893        SNHG8  ...  0.286194            -1.126985

[2 rows x 10 columns]
Background DataFrame (nrow: 110):
           Ensembl Gene_Symbols         p  ...       BHQ  Bonf  Log_AD_CTL_ampRatio
0  ENSG00000049246         PER3  0.252187  ...  0.777778   1.0             0.326548
1  ENSG00000130775      THEMIS2  0.705588  ...  0.949272   1.0             0.371321
2  ENSG00000142920        AZIN2  0.356249  ...  0.777778   1.0             0.223556
3  ENSG00000132122       SPATA6  0.240131  ...  0.777778   1.0             0.237122
4  ENSG00000162645         GBP2  0.370574  ...  0.777778   1.0             0.647551

[5 rows x 10 columns]
Directory "EnrichR_results/EnrichR_DRcyclersCyclingBHQ1AR33DRBHQ2_BCKGRND-diffrhythmsCyclingBHQ1AmpRatio33" created.

/Users/henryhollis/Box Sync/Henry_stuff/AD_project/scROSMAP/Rscripts/automatic_downstream_analysis/Python_EnrichR_for_AnalysisPipeline.py:177: RuntimeWarning: More than 20 figures have been opened. Figures created through the pyplot interface (`matplotlib.pyplot.figure`) are retained until explicitly closed and may consume too much memory. (To control this warning, see the rcParam `figure.max_open_warning`).
  fig, axes = plt.subplots(2, 2, figsize=(15, 10))

{'lib': ['KEGG_2021_Human', 'GO_Biological_Process_2023', 'MSigDB_Hallmark_2020', 'WikiPathway_2021_Human', 'TRRUST_Transcription_Factors_2019', 'GO_Cellular_Component_2023', 'TRANSFAC_and_JASPAR_PWMs', 'Transcription_Factor_PPIs', 'TargetScan_microRNA_2017', 'miRTarBase_2017'], 'genes_list_file': 'enrichR_files/DR_gainAmpAD_CyclingBHQ1AR1_DRBHQ2.csv', 'background_list_file': 'diff_rhythms_CyclingBHQ1AmpRatio1.csv'}
Please select file containing list of genes (Symbols)
Path to genes list: enrichR_files/DR_gainAmpAD_CyclingBHQ1AR1_DRBHQ2.csv

Please select file containing list of BACKGROUND genes (Symbols)
Path to Background list: diff_rhythms_CyclingBHQ1AmpRatio1.csv
Background DataFrame (nrow: 12):
           Ensembl Gene_Symbols
0  ENSG00000136715       SAP130
1  ENSG00000127990         SGCE
2  ENSG00000164985        PSIP1
3  ENSG00000148219        ASTN2
4  ENSG00000119414        PPP6C
Background DataFrame (nrow: 3130):
           Ensembl Gene_Symbols         p  ...       BHQ  Bonf  Log_AD_CTL_ampRatio
0  ENSG00000131584        ACAP3  0.669440  ...  0.861040   1.0            -0.772190
1  ENSG00000175756     AURKAIP1  0.526738  ...  0.796164   1.0             0.185463
2  ENSG00000142609       CFAP74  0.238942  ...  0.629488   1.0            -0.349632
3  ENSG00000187730        GABRD  0.490882  ...  0.786366   1.0             0.532005
4  ENSG00000158109       TPRG1L  0.834309  ...  0.933889   1.0            -0.279667

[5 rows x 10 columns]
Directory "EnrichR_results/EnrichR_DRgainAmpADCyclingBHQ1AR1DRBHQ2_BCKGRND-diffrhythmsCyclingBHQ1AmpRatio1" created.
Saved KEGG_2021_Human results to file
Saved GO_Biological_Process_2023 results to file
Saved MSigDB_Hallmark_2020 results to file
Saved WikiPathway_2021_Human results to file
Saved TRRUST_Transcription_Factors_2019 results to file
Saved GO_Cellular_Component_2023 results to file
Saved TRANSFAC_and_JASPAR_PWMs results to file
Saved Transcription_Factor_PPIs results to file
Saved TargetScan_microRNA_2017 results to file
Saved miRTarBase_2017 results to file
{'lib': ['KEGG_2021_Human', 'GO_Biological_Process_2023', 'MSigDB_Hallmark_2020', 'WikiPathway_2021_Human', 'TRRUST_Transcription_Factors_2019', 'GO_Cellular_Component_2023', 'TRANSFAC_and_JASPAR_PWMs', 'Transcription_Factor_PPIs', 'TargetScan_microRNA_2017', 'miRTarBase_2017'], 'genes_list_file': 'enrichR_files/DR_gainAmpAD_CyclingBHQ1AR25_DRBHQ2.csv', 'background_list_file': 'diff_rhythms_CyclingBHQ1AmpRatio25.csv'}
Please select file containing list of genes (Symbols)
Path to genes list: enrichR_files/DR_gainAmpAD_CyclingBHQ1AR25_DRBHQ2.csv

Please select file containing list of BACKGROUND genes (Symbols)
Path to Background list: diff_rhythms_CyclingBHQ1AmpRatio25.csv
Background DataFrame (nrow: 4):
           Ensembl Gene_Symbols
0  ENSG00000136261         BZW2
1  ENSG00000148219        ASTN2
2  ENSG00000095261        PSMD5
3  ENSG00000140470     ADAMTS17
Background DataFrame (nrow: 282):
           Ensembl Gene_Symbols         p  ...       BHQ  Bonf  Log_AD_CTL_ampRatio
0  ENSG00000097021        ACOT7  0.009033  ...  0.282688   1.0             1.408467
1  ENSG00000171680      PLEKHG5  0.086548  ...  0.488133   1.0             0.943269
2  ENSG00000049246         PER3  0.252187  ...  0.700387   1.0             0.326548
3  ENSG00000028137     TNFRSF1B  0.357533  ...  0.741148   1.0             0.499864
4  ENSG00000173436      MICOS10  0.628646  ...  0.864771   1.0             0.217262

[5 rows x 10 columns]
Directory "EnrichR_results/EnrichR_DRgainAmpADCyclingBHQ1AR25DRBHQ2_BCKGRND-diffrhythmsCyclingBHQ1AmpRatio25" created.
{'lib': ['KEGG_2021_Human', 'GO_Biological_Process_2023', 'MSigDB_Hallmark_2020', 'WikiPathway_2021_Human', 'TRRUST_Transcription_Factors_2019', 'GO_Cellular_Component_2023', 'TRANSFAC_and_JASPAR_PWMs', 'Transcription_Factor_PPIs', 'TargetScan_microRNA_2017', 'miRTarBase_2017'], 'genes_list_file': 'enrichR_files/DR_gainAmpAD_CyclingBHQ1AR33_DRBHQ2.csv', 'background_list_file': 'diff_rhythms_CyclingBHQ1AmpRatio33.csv'}
Please select file containing list of genes (Symbols)
Path to genes list: enrichR_files/DR_gainAmpAD_CyclingBHQ1AR33_DRBHQ2.csv

Please select file containing list of BACKGROUND genes (Symbols)
Path to Background list: diff_rhythms_CyclingBHQ1AmpRatio33.csv
Background DataFrame (nrow: 1):
           Ensembl Gene_Symbols
0  ENSG00000140470     ADAMTS17
Background DataFrame (nrow: 110):
           Ensembl Gene_Symbols         p  ...       BHQ  Bonf  Log_AD_CTL_ampRatio
0  ENSG00000049246         PER3  0.252187  ...  0.777778   1.0             0.326548
1  ENSG00000130775      THEMIS2  0.705588  ...  0.949272   1.0             0.371321
2  ENSG00000142920        AZIN2  0.356249  ...  0.777778   1.0             0.223556
3  ENSG00000132122       SPATA6  0.240131  ...  0.777778   1.0             0.237122
4  ENSG00000162645         GBP2  0.370574  ...  0.777778   1.0             0.647551

[5 rows x 10 columns]
Directory "EnrichR_results/EnrichR_DRgainAmpADCyclingBHQ1AR33DRBHQ2_BCKGRND-diffrhythmsCyclingBHQ1AmpRatio33" created.
{'lib': ['KEGG_2021_Human', 'GO_Biological_Process_2023', 'MSigDB_Hallmark_2020', 'WikiPathway_2021_Human', 'TRRUST_Transcription_Factors_2019', 'GO_Cellular_Component_2023', 'TRANSFAC_and_JASPAR_PWMs', 'Transcription_Factor_PPIs', 'TargetScan_microRNA_2017', 'miRTarBase_2017'], 'genes_list_file': 'enrichR_files/DR_lostAmpAD_CyclingBHQ1AR1_DRBHQ2.csv', 'background_list_file': 'diff_rhythms_CyclingBHQ1AmpRatio1.csv'}
Please select file containing list of genes (Symbols)
Path to genes list: enrichR_files/DR_lostAmpAD_CyclingBHQ1AR1_DRBHQ2.csv

Please select file containing list of BACKGROUND genes (Symbols)
Path to Background list: diff_rhythms_CyclingBHQ1AmpRatio1.csv
Background DataFrame (nrow: 0):
Empty DataFrame
Columns: [Ensembl, Gene_Symbols]
Index: []
Background DataFrame (nrow: 3130):
           Ensembl Gene_Symbols         p  ...       BHQ  Bonf  Log_AD_CTL_ampRatio
0  ENSG00000131584        ACAP3  0.669440  ...  0.861040   1.0            -0.772190
1  ENSG00000175756     AURKAIP1  0.526738  ...  0.796164   1.0             0.185463
2  ENSG00000142609       CFAP74  0.238942  ...  0.629488   1.0            -0.349632
3  ENSG00000187730        GABRD  0.490882  ...  0.786366   1.0             0.532005
4  ENSG00000158109       TPRG1L  0.834309  ...  0.933889   1.0            -0.279667

[5 rows x 10 columns]
Directory "EnrichR_results/EnrichR_DRlostAmpADCyclingBHQ1AR1DRBHQ2_BCKGRND-diffrhythmsCyclingBHQ1AmpRatio1" created.
{'lib': ['KEGG_2021_Human', 'GO_Biological_Process_2023', 'MSigDB_Hallmark_2020', 'WikiPathway_2021_Human', 'TRRUST_Transcription_Factors_2019', 'GO_Cellular_Component_2023', 'TRANSFAC_and_JASPAR_PWMs', 'Transcription_Factor_PPIs', 'TargetScan_microRNA_2017', 'miRTarBase_2017'], 'genes_list_file': 'enrichR_files/DR_lostAmpAD_CyclingBHQ1AR25_DRBHQ2.csv', 'background_list_file': 'diff_rhythms_CyclingBHQ1AmpRatio25.csv'}
Please select file containing list of genes (Symbols)
Path to genes list: enrichR_files/DR_lostAmpAD_CyclingBHQ1AR25_DRBHQ2.csv

Please select file containing list of BACKGROUND genes (Symbols)
Path to Background list: diff_rhythms_CyclingBHQ1AmpRatio25.csv
Background DataFrame (nrow: 3):
           Ensembl Gene_Symbols
0  ENSG00000158258       CLSTN2
1  ENSG00000269893        SNHG8
2  ENSG00000134419       RPS15A
Background DataFrame (nrow: 282):
           Ensembl Gene_Symbols         p  ...       BHQ  Bonf  Log_AD_CTL_ampRatio
0  ENSG00000097021        ACOT7  0.009033  ...  0.282688   1.0             1.408467
1  ENSG00000171680      PLEKHG5  0.086548  ...  0.488133   1.0             0.943269
2  ENSG00000049246         PER3  0.252187  ...  0.700387   1.0             0.326548
3  ENSG00000028137     TNFRSF1B  0.357533  ...  0.741148   1.0             0.499864
4  ENSG00000173436      MICOS10  0.628646  ...  0.864771   1.0             0.217262

[5 rows x 10 columns]
Directory "EnrichR_results/EnrichR_DRlostAmpADCyclingBHQ1AR25DRBHQ2_BCKGRND-diffrhythmsCyclingBHQ1AmpRatio25" created.
Saved KEGG_2021_Human results to file
Saved GO_Biological_Process_2023 results to file
{'lib': ['KEGG_2021_Human', 'GO_Biological_Process_2023', 'MSigDB_Hallmark_2020', 'WikiPathway_2021_Human', 'TRRUST_Transcription_Factors_2019', 'GO_Cellular_Component_2023', 'TRANSFAC_and_JASPAR_PWMs', 'Transcription_Factor_PPIs', 'TargetScan_microRNA_2017', 'miRTarBase_2017'], 'genes_list_file': 'enrichR_files/DR_lostAmpAD_CyclingBHQ1AR33_DRBHQ2.csv', 'background_list_file': 'diff_rhythms_CyclingBHQ1AmpRatio33.csv'}
Please select file containing list of genes (Symbols)
Path to genes list: enrichR_files/DR_lostAmpAD_CyclingBHQ1AR33_DRBHQ2.csv

Please select file containing list of BACKGROUND genes (Symbols)
Path to Background list: diff_rhythms_CyclingBHQ1AmpRatio33.csv
Background DataFrame (nrow: 1):
           Ensembl Gene_Symbols
0  ENSG00000269893        SNHG8
Background DataFrame (nrow: 110):
           Ensembl Gene_Symbols         p  ...       BHQ  Bonf  Log_AD_CTL_ampRatio
0  ENSG00000049246         PER3  0.252187  ...  0.777778   1.0             0.326548
1  ENSG00000130775      THEMIS2  0.705588  ...  0.949272   1.0             0.371321
2  ENSG00000142920        AZIN2  0.356249  ...  0.777778   1.0             0.223556
3  ENSG00000132122       SPATA6  0.240131  ...  0.777778   1.0             0.237122
4  ENSG00000162645         GBP2  0.370574  ...  0.777778   1.0             0.647551

[5 rows x 10 columns]
Directory "EnrichR_results/EnrichR_DRlostAmpADCyclingBHQ1AR33DRBHQ2_BCKGRND-diffrhythmsCyclingBHQ1AmpRatio33" created.

/Users/henryhollis/Box Sync/Henry_stuff/AD_project/scROSMAP/Rscripts/automatic_downstream_analysis/Python_EnrichR_for_AnalysisPipeline.py:177: RuntimeWarning: More than 20 figures have been opened. Figures created through the pyplot interface (`matplotlib.pyplot.figure`) are retained until explicitly closed and may consume too much memory. (To control this warning, see the rcParam `figure.max_open_warning`).
  fig, axes = plt.subplots(2, 2, figsize=(15, 10))

{'lib': ['KEGG_2021_Human', 'GO_Biological_Process_2023', 'MSigDB_Hallmark_2020', 'WikiPathway_2021_Human', 'TRRUST_Transcription_Factors_2019', 'GO_Cellular_Component_2023', 'TRANSFAC_and_JASPAR_PWMs', 'Transcription_Factor_PPIs', 'TargetScan_microRNA_2017', 'miRTarBase_2017'], 'genes_list_file': 'enrichR_files/DR_cyclers_mthd2_CyclingBHQ1AR1_DRBHQ2.csv', 'background_list_file': 'diff_rhythms_method2_CyclingBHQ1AmpRatio1.csv'}
Please select file containing list of genes (Symbols)
Path to genes list: enrichR_files/DR_cyclers_mthd2_CyclingBHQ1AR1_DRBHQ2.csv

Please select file containing list of BACKGROUND genes (Symbols)
Path to Background list: diff_rhythms_method2_CyclingBHQ1AmpRatio1.csv
Background DataFrame (nrow: 12):
           Ensembl Gene_Symbols  ...      Bonf  Log_AD_CTL_ampRatio
0  ENSG00000136715       SAP130  ...  0.416290             0.238614
1  ENSG00000127990         SGCE  ...  1.000000             0.866906
2  ENSG00000164985        PSIP1  ...  0.975270             2.777088
3  ENSG00000119414        PPP6C  ...  1.000000             1.002309
4  ENSG00000148606       POLR3A  ...  0.506969             0.654147

[5 rows x 10 columns]
Background DataFrame (nrow: 3184):
           Ensembl Gene_Symbols         p  ...       BHQ  Bonf  Log_AD_CTL_ampRatio
0  ENSG00000078808         SDF4  0.754184  ...  0.904053   1.0             0.300964
1  ENSG00000175756     AURKAIP1  0.526738  ...  0.806833   1.0             0.185463
2  ENSG00000242485       MRPL20  0.033929  ...  0.387206   1.0             0.258299
3  ENSG00000142609       CFAP74  0.238942  ...  0.638216   1.0            -0.349632
4  ENSG00000187730        GABRD  0.490882  ...  0.796315   1.0             0.532005

[5 rows x 10 columns]
Directory "EnrichR_results/EnrichR_DRcyclersmthd2CyclingBHQ1AR1DRBHQ2_BCKGRND-diffrhythmsmethod2CyclingBHQ1AmpRatio1" created.
Saved KEGG_2021_Human results to file
Saved GO_Biological_Process_2023 results to file
Saved MSigDB_Hallmark_2020 results to file
Saved WikiPathway_2021_Human results to file
Saved TRRUST_Transcription_Factors_2019 results to file
Saved GO_Cellular_Component_2023 results to file
Saved TRANSFAC_and_JASPAR_PWMs results to file
Saved Transcription_Factor_PPIs results to file
Saved TargetScan_microRNA_2017 results to file
Saved miRTarBase_2017 results to file
{'lib': ['KEGG_2021_Human', 'GO_Biological_Process_2023', 'MSigDB_Hallmark_2020', 'WikiPathway_2021_Human', 'TRRUST_Transcription_Factors_2019', 'GO_Cellular_Component_2023', 'TRANSFAC_and_JASPAR_PWMs', 'Transcription_Factor_PPIs', 'TargetScan_microRNA_2017', 'miRTarBase_2017'], 'genes_list_file': 'enrichR_files/DR_cyclers_mthd2_CyclingBHQ1AR25_DRBHQ2.csv', 'background_list_file': 'diff_rhythms_method2_CyclingBHQ1AmpRatio25.csv'}
Please select file containing list of genes (Symbols)
Path to genes list: enrichR_files/DR_cyclers_mthd2_CyclingBHQ1AR25_DRBHQ2.csv

Please select file containing list of BACKGROUND genes (Symbols)
Path to Background list: diff_rhythms_method2_CyclingBHQ1AmpRatio25.csv
Background DataFrame (nrow: 9):
           Ensembl Gene_Symbols  ...      Bonf  Log_AD_CTL_ampRatio
0  ENSG00000127990         SGCE  ...  0.097682             0.866906
1  ENSG00000148219        ASTN2  ...  0.135941             1.276032
2  ENSG00000140470     ADAMTS17  ...  0.070903             2.177640
3  ENSG00000095261        PSMD5  ...  0.393666             1.203439
4  ENSG00000134419       RPS15A  ...  0.460067            -1.276418

[5 rows x 10 columns]
Background DataFrame (nrow: 301):
           Ensembl Gene_Symbols         p  ...       BHQ  Bonf  Log_AD_CTL_ampRatio
0  ENSG00000097021        ACOT7  0.009033  ...  0.247176   1.0             1.408467
1  ENSG00000171680      PLEKHG5  0.086548  ...  0.482427   1.0             0.943269
2  ENSG00000049246         PER3  0.252187  ...  0.706044   1.0             0.326548
3  ENSG00000028137     TNFRSF1B  0.357533  ...  0.758794   1.0             0.499864
4  ENSG00000173436      MICOS10  0.628646  ...  0.867992   1.0             0.217262

[5 rows x 10 columns]
Directory "EnrichR_results/EnrichR_DRcyclersmthd2CyclingBHQ1AR25DRBHQ2_BCKGRND-diffrhythmsmethod2CyclingBHQ1AmpRatio25" created.
Saved KEGG_2021_Human results to file
Saved GO_Biological_Process_2023 results to file
Saved MSigDB_Hallmark_2020 results to file
Saved WikiPathway_2021_Human results to file

/Users/henryhollis/Box Sync/Henry_stuff/AD_project/scROSMAP/Rscripts/automatic_downstream_analysis/Python_EnrichR_for_AnalysisPipeline.py:177: RuntimeWarning: More than 20 figures have been opened. Figures created through the pyplot interface (`matplotlib.pyplot.figure`) are retained until explicitly closed and may consume too much memory. (To control this warning, see the rcParam `figure.max_open_warning`).
  fig, axes = plt.subplots(2, 2, figsize=(15, 10))

{'lib': ['KEGG_2021_Human', 'GO_Biological_Process_2023', 'MSigDB_Hallmark_2020', 'WikiPathway_2021_Human', 'TRRUST_Transcription_Factors_2019', 'GO_Cellular_Component_2023', 'TRANSFAC_and_JASPAR_PWMs', 'Transcription_Factor_PPIs', 'TargetScan_microRNA_2017', 'miRTarBase_2017'], 'genes_list_file': 'enrichR_files/DR_mthd2_gainAmpAD_CyclingBHQ1AR1_DRBHQ2.csv', 'background_list_file': 'diff_rhythms_method2_CyclingBHQ1AmpRatio1.csv'}
Please select file containing list of genes (Symbols)
Path to genes list: enrichR_files/DR_mthd2_gainAmpAD_CyclingBHQ1AR1_DRBHQ2.csv

Please select file containing list of BACKGROUND genes (Symbols)
Path to Background list: diff_rhythms_method2_CyclingBHQ1AmpRatio1.csv
Background DataFrame (nrow: 12):
           Ensembl Gene_Symbols
0  ENSG00000136715       SAP130
1  ENSG00000127990         SGCE
2  ENSG00000164985        PSIP1
3  ENSG00000148219        ASTN2
4  ENSG00000119414        PPP6C
Background DataFrame (nrow: 3184):
           Ensembl Gene_Symbols         p  ...       BHQ  Bonf  Log_AD_CTL_ampRatio
0  ENSG00000078808         SDF4  0.754184  ...  0.904053   1.0             0.300964
1  ENSG00000175756     AURKAIP1  0.526738  ...  0.806833   1.0             0.185463
2  ENSG00000242485       MRPL20  0.033929  ...  0.387206   1.0             0.258299
3  ENSG00000142609       CFAP74  0.238942  ...  0.638216   1.0            -0.349632
4  ENSG00000187730        GABRD  0.490882  ...  0.796315   1.0             0.532005

[5 rows x 10 columns]
Directory "EnrichR_results/EnrichR_DRmthd2gainAmpADCyclingBHQ1AR1DRBHQ2_BCKGRND-diffrhythmsmethod2CyclingBHQ1AmpRatio1" created.
Saved KEGG_2021_Human results to file
Saved GO_Biological_Process_2023 results to file
Saved MSigDB_Hallmark_2020 results to file
Saved WikiPathway_2021_Human results to file
Saved TRRUST_Transcription_Factors_2019 results to file
Saved GO_Cellular_Component_2023 results to file
Saved TRANSFAC_and_JASPAR_PWMs results to file
Saved Transcription_Factor_PPIs results to file
Saved TargetScan_microRNA_2017 results to file
Saved miRTarBase_2017 results to file
{'lib': ['KEGG_2021_Human', 'GO_Biological_Process_2023', 'MSigDB_Hallmark_2020', 'WikiPathway_2021_Human', 'TRRUST_Transcription_Factors_2019', 'GO_Cellular_Component_2023', 'TRANSFAC_and_JASPAR_PWMs', 'Transcription_Factor_PPIs', 'TargetScan_microRNA_2017', 'miRTarBase_2017'], 'genes_list_file': 'enrichR_files/DR_mthd2_gainAmpAD_CyclingBHQ1AR25_DRBHQ2.csv', 'background_list_file': 'diff_rhythms_method2_CyclingBHQ1AmpRatio25.csv'}
Please select file containing list of genes (Symbols)
Path to genes list: enrichR_files/DR_mthd2_gainAmpAD_CyclingBHQ1AR25_DRBHQ2.csv

Please select file containing list of BACKGROUND genes (Symbols)
Path to Background list: diff_rhythms_method2_CyclingBHQ1AmpRatio25.csv
Background DataFrame (nrow: 5):
           Ensembl Gene_Symbols
0  ENSG00000136261         BZW2
1  ENSG00000127990         SGCE
2  ENSG00000148219        ASTN2
3  ENSG00000095261        PSMD5
4  ENSG00000140470     ADAMTS17
Background DataFrame (nrow: 301):
           Ensembl Gene_Symbols         p  ...       BHQ  Bonf  Log_AD_CTL_ampRatio
0  ENSG00000097021        ACOT7  0.009033  ...  0.247176   1.0             1.408467
1  ENSG00000171680      PLEKHG5  0.086548  ...  0.482427   1.0             0.943269
2  ENSG00000049246         PER3  0.252187  ...  0.706044   1.0             0.326548
3  ENSG00000028137     TNFRSF1B  0.357533  ...  0.758794   1.0             0.499864
4  ENSG00000173436      MICOS10  0.628646  ...  0.867992   1.0             0.217262

[5 rows x 10 columns]
Directory "EnrichR_results/EnrichR_DRmthd2gainAmpADCyclingBHQ1AR25DRBHQ2_BCKGRND-diffrhythmsmethod2CyclingBHQ1AmpRatio25" created.
{'lib': ['KEGG_2021_Human', 'GO_Biological_Process_2023', 'MSigDB_Hallmark_2020', 'WikiPathway_2021_Human', 'TRRUST_Transcription_Factors_2019', 'GO_Cellular_Component_2023', 'TRANSFAC_and_JASPAR_PWMs', 'Transcription_Factor_PPIs', 'TargetScan_microRNA_2017', 'miRTarBase_2017'], 'genes_list_file': 'enrichR_files/DR_mthd2_lostAmpAD_CyclingBHQ1AR1_DRBHQ2.csv', 'background_list_file': 'diff_rhythms_method2_CyclingBHQ1AmpRatio1.csv'}
Please select file containing list of genes (Symbols)
Path to genes list: enrichR_files/DR_mthd2_lostAmpAD_CyclingBHQ1AR1_DRBHQ2.csv

Please select file containing list of BACKGROUND genes (Symbols)
Path to Background list: diff_rhythms_method2_CyclingBHQ1AmpRatio1.csv
Background DataFrame (nrow: 0):
Empty DataFrame
Columns: [Ensembl, Gene_Symbols]
Index: []
Background DataFrame (nrow: 3184):
           Ensembl Gene_Symbols         p  ...       BHQ  Bonf  Log_AD_CTL_ampRatio
0  ENSG00000078808         SDF4  0.754184  ...  0.904053   1.0             0.300964
1  ENSG00000175756     AURKAIP1  0.526738  ...  0.806833   1.0             0.185463
2  ENSG00000242485       MRPL20  0.033929  ...  0.387206   1.0             0.258299
3  ENSG00000142609       CFAP74  0.238942  ...  0.638216   1.0            -0.349632
4  ENSG00000187730        GABRD  0.490882  ...  0.796315   1.0             0.532005

[5 rows x 10 columns]
Directory "EnrichR_results/EnrichR_DRmthd2lostAmpADCyclingBHQ1AR1DRBHQ2_BCKGRND-diffrhythmsmethod2CyclingBHQ1AmpRatio1" created.
{'lib': ['KEGG_2021_Human', 'GO_Biological_Process_2023', 'MSigDB_Hallmark_2020', 'WikiPathway_2021_Human', 'TRRUST_Transcription_Factors_2019', 'GO_Cellular_Component_2023', 'TRANSFAC_and_JASPAR_PWMs', 'Transcription_Factor_PPIs', 'TargetScan_microRNA_2017', 'miRTarBase_2017'], 'genes_list_file': 'enrichR_files/DR_mthd2_lostAmpAD_CyclingBHQ1AR25_DRBHQ2.csv', 'background_list_file': 'diff_rhythms_method2_CyclingBHQ1AmpRatio25.csv'}
Please select file containing list of genes (Symbols)
Path to genes list: enrichR_files/DR_mthd2_lostAmpAD_CyclingBHQ1AR25_DRBHQ2.csv

Please select file containing list of BACKGROUND genes (Symbols)
Path to Background list: diff_rhythms_method2_CyclingBHQ1AmpRatio25.csv
Background DataFrame (nrow: 4):
           Ensembl Gene_Symbols
0  ENSG00000158258       CLSTN2
1  ENSG00000269893        SNHG8
2  ENSG00000196683        TOMM7
3  ENSG00000134419       RPS15A
Background DataFrame (nrow: 301):
           Ensembl Gene_Symbols         p  ...       BHQ  Bonf  Log_AD_CTL_ampRatio
0  ENSG00000097021        ACOT7  0.009033  ...  0.247176   1.0             1.408467
1  ENSG00000171680      PLEKHG5  0.086548  ...  0.482427   1.0             0.943269
2  ENSG00000049246         PER3  0.252187  ...  0.706044   1.0             0.326548
3  ENSG00000028137     TNFRSF1B  0.357533  ...  0.758794   1.0             0.499864
4  ENSG00000173436      MICOS10  0.628646  ...  0.867992   1.0             0.217262

[5 rows x 10 columns]
Directory "EnrichR_results/EnrichR_DRmthd2lostAmpADCyclingBHQ1AR25DRBHQ2_BCKGRND-diffrhythmsmethod2CyclingBHQ1AmpRatio25" created.
Saved KEGG_2021_Human results to file
Saved GO_Biological_Process_2023 results to file
         DR_cyclers_CyclingBHQ1AR1_DRBHQ2.csv         DR_cyclers_CyclingBHQ1AR25_DRBHQ2.csv         DR_cyclers_CyclingBHQ1AR33_DRBHQ2.csv 
                                            0                                             0                                             0 
       DR_gainAmpAD_CyclingBHQ1AR1_DRBHQ2.csv       DR_gainAmpAD_CyclingBHQ1AR25_DRBHQ2.csv       DR_gainAmpAD_CyclingBHQ1AR33_DRBHQ2.csv 
                                            0                                             0                                             0 
       DR_lostAmpAD_CyclingBHQ1AR1_DRBHQ2.csv       DR_lostAmpAD_CyclingBHQ1AR25_DRBHQ2.csv       DR_lostAmpAD_CyclingBHQ1AR33_DRBHQ2.csv 
                                            0                                             0                                             0 
   DR_cyclers_mthd2_CyclingBHQ1AR1_DRBHQ2.csv   DR_cyclers_mthd2_CyclingBHQ1AR25_DRBHQ2.csv  DR_mthd2_gainAmpAD_CyclingBHQ1AR1_DRBHQ2.csv 
                                            0                                             0                                             0 
DR_mthd2_gainAmpAD_CyclingBHQ1AR25_DRBHQ2.csv  DR_mthd2_lostAmpAD_CyclingBHQ1AR1_DRBHQ2.csv DR_mthd2_lostAmpAD_CyclingBHQ1AR25_DRBHQ2.csv 
                                            0                                             0                                             0 

#AD severity
system(paste0("python3 ", path_no_space, "/Python_EnrichR_for_AnalysisPipeline.py -g \"enrichR_files/strong_cogdx_diffs_AR25.csv\" -b \"enrichR_files/AD_cyclers_AR25BHQ05.csv\""))

{'lib': ['KEGG_2021_Human', 'GO_Biological_Process_2023', 'MSigDB_Hallmark_2020', 'WikiPathway_2021_Human', 'TRRUST_Transcription_Factors_2019', 'GO_Cellular_Component_2023', 'TRANSFAC_and_JASPAR_PWMs', 'Transcription_Factor_PPIs', 'TargetScan_microRNA_2017', 'miRTarBase_2017'], 'genes_list_file': 'enrichR_files/strong_cogdx_diffs_AR25.csv', 'background_list_file': 'enrichR_files/AD_cyclers_AR25BHQ05.csv'}
Please select file containing list of genes (Symbols)
Path to genes list: enrichR_files/strong_cogdx_diffs_AR25.csv

Please select file containing list of BACKGROUND genes (Symbols)
Path to Background list: enrichR_files/AD_cyclers_AR25BHQ05.csv
Background DataFrame (nrow: 0):
Empty DataFrame
Columns: [Ensembl, Gene_Symbols]
Index: []
File not found.

Traceback (most recent call last):
  File "/Users/henryhollis/Box Sync/Henry_stuff/AD_project/scROSMAP/Rscripts/automatic_downstream_analysis/Python_EnrichR_for_AnalysisPipeline.py", line 90, in <module>
    background = list(background_df['Gene_Symbols'])
TypeError: 'NoneType' object is not subscriptable

FGSEA

path = getwd()
setwd(path_to_cyclops_ordering)

Warning: The working directory was changed to /Users/henryhollis/Box Sync/Henry_stuff/AD_project/human_data/Cyclops_folders/training_output/scROSMAP/cogdx_controls/wAD/ExcitatoryNeurons/TMMs_w_batch/Exc3and5_FiltByEdgeRDefault_fixedipBulkChenZhang_condAndBatchCovs_04ContrVar4EGdefault_noTransferFit_Feb5Redo inside a notebook chunk. The working directory will be reset when the chunk is finished running. Use the knitr root.dir option in the setup chunk to change the working directory for notebook chunks.

if (!(dir.exists("downstream_output/fGSEA"))){
  dir.create("downstream_output/fGSEA")
  dir.create(paste("downstream_output", "fGSEA", "rnk_files", sep = "/"))
  dir.create(paste("downstream_output" , "fGSEA", "fGSEA_results", sep = "/"))
}

isCyclingBHQCutoff_str = str_extract(as.character(isCyclingBHQCutoff), "(?<=\\.)\\d+")

#create rnk files
write_rnks(path_to_cyclops_ordering, isCyclingBHQCutoff_str)

[1] "Creating rnk files for fGSEA."

#path of parent folder
setwd(path)

#Pathways, downloaded from MsigDB
pathways <- c(gmtPathways("./MsigDB_gmts_for_GSEA/c2.cp.kegg.v2023.1.Hs.symbols.gmt"), gmtPathways("./MsigDB_gmts_for_GSEA/h.all.v2023.1.Hs.symbols.gmt"))


gene_remapping_dict = read.delim("./MsigDB_gmts_for_GSEA/Human_Gene_Symbol_with_Remapping_MSigDB.v2023.1.Hs.chip", sep = '\t')

setwd(paste(path_to_cyclops_ordering,"downstream_output", "fGSEA", "rnk_files", sep = "/"))

rnk_files = list.files(pattern = ".rnk")


if(!(dir.exists(paste(path_to_cyclops_ordering, "downstream_output","fGSEA","fGSEA_results", sep = "/")))){
  dir.create( paste(path_to_cyclops_ordering,"downstream_output", "fGSEA","fGSEA_results", sep = "/") )
  dir.create( paste(path_to_cyclops_ordering,"downstream_output", "fGSEA","fGSEA_results", "plots",sep = "/"))
}

run_fgsea(rnk_files, gene_remapping_dict, pathways)

[1] "../fGSEA_results/AD_cyclers_minusLogPRanked.csv"
[1] "Used pos fGSEA score type"

Saving 7 x 7 in image

[1] "../fGSEA_results/CTL_cyclers_minusLogPRanked.csv"
[1] "Used pos fGSEA score type"
[1] "../fGSEA_results/differential_mesor_all_genes_minusLogPRanked.csv"
[1] "Used pos fGSEA score type"
[1] "../fGSEA_results/DRgenesAmpRatio1_Log(AD-CTL)ranked.csv"
[1] "Used std fGSEA score type"
[1] "../fGSEA_results/DRgenesAmpRatio1_minusLogPRanked.csv"
[1] "Used pos fGSEA score type"
[1] "../fGSEA_results/DRgenesAmpRatio25_Log(AD-CTL)ranked_method2.csv"
[1] "Used std fGSEA score type"
[1] "../fGSEA_results/DRgenesAmpRatio25_Log(AD-CTL)ranked.csv"
[1] "Used std fGSEA score type"
[1] "../fGSEA_results/DRgenesAmpRatio25_minusLogPRanked_method2.csv"
[1] "Used pos fGSEA score type"
[1] "../fGSEA_results/DRgenesAmpRatio25_minusLogPRanked.csv"
[1] "Used pos fGSEA score type"
                                           AD_cyclers_minusLogPRanked.rnk 
                  "../fGSEA_results/plots/AD_cyclers_minusLogPRanked.png" 
                                          CTL_cyclers_minusLogPRanked.rnk 
                 "../fGSEA_results/plots/CTL_cyclers_minusLogPRanked.png" 
                         differential_mesor_all_genes_minusLogPRanked.rnk 
"../fGSEA_results/plots/differential_mesor_all_genes_minusLogPRanked.png" 
                                   DRgenesAmpRatio1_Log(AD-CTL)ranked.rnk 
          "../fGSEA_results/plots/DRgenesAmpRatio1_Log(AD-CTL)ranked.png" 
                                     DRgenesAmpRatio1_minusLogPRanked.rnk 
            "../fGSEA_results/plots/DRgenesAmpRatio1_minusLogPRanked.png" 
                          DRgenesAmpRatio25_Log(AD-CTL)ranked_method2.rnk 
 "../fGSEA_results/plots/DRgenesAmpRatio25_Log(AD-CTL)ranked_method2.png" 
                                  DRgenesAmpRatio25_Log(AD-CTL)ranked.rnk 
         "../fGSEA_results/plots/DRgenesAmpRatio25_Log(AD-CTL)ranked.png" 
                            DRgenesAmpRatio25_minusLogPRanked_method2.rnk 
   "../fGSEA_results/plots/DRgenesAmpRatio25_minusLogPRanked_method2.png" 
                                    DRgenesAmpRatio25_minusLogPRanked.rnk 
           "../fGSEA_results/plots/DRgenesAmpRatio25_minusLogPRanked.png" 

Run fgsea on sleep related pathways from https://doi.org/10.1152/physiolgenomics.00275.2006

sleep_wake_pathways = gmtPathways("./MsigDB_gmts_for_GSEA/sleep_wake_pathways_w_all_human_aliases_filtered_to_background_genes.gmt")

if(!(dir.exists(paste(path_to_cyclops_ordering, "downstream_output","fGSEA","fGSEA_results_sleep_wake", sep = "/")))){
    dir.create( paste(path_to_cyclops_ordering,"downstream_output", "fGSEA","fGSEA_results_sleep_wake", sep = "/") )
    dir.create( paste(path_to_cyclops_ordering,"downstream_output", "fGSEA","fGSEA_results_sleep_wake", "plots", sep = "/") )
}
setwd(paste(path_to_cyclops_ordering,"downstream_output", "fGSEA", "rnk_files", sep = "/"))

Warning: The working directory was changed to /Users/henryhollis/Box Sync/Henry_stuff/AD_project/human_data/Cyclops_folders/training_output/scROSMAP/cogdx_controls/wAD/ExcitatoryNeurons/TMMs_w_batch/Exc3and5_FiltByEdgeRDefault_fixedipBulkChenZhang_condAndBatchCovs_04ContrVar4EGdefault_noTransferFit_Feb5Redo/downstream_output/fGSEA/rnk_files inside a notebook chunk. The working directory will be reset when the chunk is finished running. Use the knitr root.dir option in the setup chunk to change the working directory for notebook chunks.

run_fgsea(rnk_files, gene_remapping_dict, sleep_wake_pathways, dir = "fGSEA_results_sleep_wake", max_size = 10000)

[1] "../fGSEA_results_sleep_wake/AD_cyclers_minusLogPRanked.csv"
[1] "Used pos fGSEA score type"

Saving 7 x 7 in image

[1] "../fGSEA_results_sleep_wake/CTL_cyclers_minusLogPRanked.csv"
[1] "Used pos fGSEA score type"
[1] "../fGSEA_results_sleep_wake/differential_mesor_all_genes_minusLogPRanked.csv"
[1] "Used pos fGSEA score type"
[1] "../fGSEA_results_sleep_wake/DRgenesAmpRatio1_Log(AD-CTL)ranked.csv"
[1] "Used std fGSEA score type"
[1] "../fGSEA_results_sleep_wake/DRgenesAmpRatio1_minusLogPRanked.csv"
[1] "Used pos fGSEA score type"
[1] "../fGSEA_results_sleep_wake/DRgenesAmpRatio25_Log(AD-CTL)ranked_method2.csv"
[1] "Used std fGSEA score type"
[1] "../fGSEA_results_sleep_wake/DRgenesAmpRatio25_Log(AD-CTL)ranked.csv"
[1] "Used std fGSEA score type"
[1] "../fGSEA_results_sleep_wake/DRgenesAmpRatio25_minusLogPRanked_method2.csv"
[1] "Used pos fGSEA score type"
[1] "../fGSEA_results_sleep_wake/DRgenesAmpRatio25_minusLogPRanked.csv"
[1] "Used pos fGSEA score type"
                                                      AD_cyclers_minusLogPRanked.rnk 
                  "../fGSEA_results_sleep_wake/plots/AD_cyclers_minusLogPRanked.png" 
                                                     CTL_cyclers_minusLogPRanked.rnk 
                 "../fGSEA_results_sleep_wake/plots/CTL_cyclers_minusLogPRanked.png" 
                                    differential_mesor_all_genes_minusLogPRanked.rnk 
"../fGSEA_results_sleep_wake/plots/differential_mesor_all_genes_minusLogPRanked.png" 
                                              DRgenesAmpRatio1_Log(AD-CTL)ranked.rnk 
          "../fGSEA_results_sleep_wake/plots/DRgenesAmpRatio1_Log(AD-CTL)ranked.png" 
                                                DRgenesAmpRatio1_minusLogPRanked.rnk 
            "../fGSEA_results_sleep_wake/plots/DRgenesAmpRatio1_minusLogPRanked.png" 
                                     DRgenesAmpRatio25_Log(AD-CTL)ranked_method2.rnk 
 "../fGSEA_results_sleep_wake/plots/DRgenesAmpRatio25_Log(AD-CTL)ranked_method2.png" 
                                             DRgenesAmpRatio25_Log(AD-CTL)ranked.rnk 
         "../fGSEA_results_sleep_wake/plots/DRgenesAmpRatio25_Log(AD-CTL)ranked.png" 
                                       DRgenesAmpRatio25_minusLogPRanked_method2.rnk 
   "../fGSEA_results_sleep_wake/plots/DRgenesAmpRatio25_minusLogPRanked_method2.png" 
                                               DRgenesAmpRatio25_minusLogPRanked.rnk 
           "../fGSEA_results_sleep_wake/plots/DRgenesAmpRatio25_minusLogPRanked.png" 

Phase Set Enrichment Analysis (PSEA)

path = getwd()
path_no_space = gsub(" ", "\\ ", path, fixed = TRUE)

# List of pathways, downloaded from MsigDB, absolute paths
psea_pathways <- c(paste0(getwd(), "/MsigDB_gmts_for_GSEA/c2.cp.kegg.v2023.1.Hs.symbols.gmt"), paste0(getwd(), "/MsigDB_gmts_for_GSEA/h.all.v2023.1.Hs.symbols.gmt"))

# make sure no spaces when calling cmd
psea_pathways = gsub(" ", "\\ ", psea_pathways, fixed = TRUE) 

# change dir into PSEA directory
setwd(paste(path_to_cyclops_ordering,"downstream_output", "PSEA_files",sep = "/"))

Warning: The working directory was changed to /Users/henryhollis/Box Sync/Henry_stuff/AD_project/human_data/Cyclops_folders/training_output/scROSMAP/cogdx_controls/wAD/ExcitatoryNeurons/TMMs_w_batch/Exc3and5_FiltByEdgeRDefault_fixedipBulkChenZhang_condAndBatchCovs_04ContrVar4EGdefault_noTransferFit_Feb5Redo/downstream_output/PSEA_files inside a notebook chunk. The working directory will be reset when the chunk is finished running. Use the knitr root.dir option in the setup chunk to change the working directory for notebook chunks.

#list PSEA input files (generated during cosinor regression/DR step)
psea_files = list.files(pattern = "*.txt")

#Save path to ordering as string for cleaning up call to system
path_to_cyclops_ordering_no_space = gsub(" ", "\\ ", path_to_cyclops_ordering, fixed = TRUE)

#dataframe where rows are arguments to system, I will apply system call across rows
arg_df = expand.grid(psea_files, psea_pathways) %>% 
      mutate(out_dirs = paste(str_extract(string = as.character(Var1),     pattern= ".+(?=\\.txt)"), basename(as.character(Var2)) %>% str_extract(pattern= ".+(?=\\.gmt)")%>% 
      gsub(pattern = "\\.", replacement = ""), sep = "_") )

#apply system call, calling PSEA jar file, using rows of arg_df as arguments
apply(arg_df, 1, function(a){
    system(paste0("java -jar ", path_no_space, "/PSEA_cmd.jar ", 
    paste0(path_to_cyclops_ordering_no_space, 
    "/downstream_output/PSEA_files/", a[[1]]), " ", a[[2]]," ",
    paste0(path_to_cyclops_ordering_no_space,
    "/downstream_output/PSEA_files/", a[[3]]), " 5 10000 pdf"))
        })

Absolute Path to gene-acrophase file: /Users/henryhollis/Box Sync/Henry_stuff/AD_project/human_data/Cyclops_folders/training_output/scROSMAP/cogdx_controls/wAD/ExcitatoryNeurons/TMMs_w_batch/Exc3and5_FiltByEdgeRDefault_fixedipBulkChenZhang_condAndBatchCovs_04ContrVar4EGdefault_noTransferFit_Feb5Redo//downstream_output/PSEA_files/PSEA_AD_cyclers_AR25BHQ1.txt
Absolute Path to gmt file: /Users/henryhollis/Box Sync/Henry_stuff/AD_project/scROSMAP/Rscripts/automatic_downstream_analysis/MsigDB_gmts_for_GSEA/c2.cp.kegg.v2023.1.Hs.symbols.gmt
Absolute Path to output folder: /Users/henryhollis/Box Sync/Henry_stuff/AD_project/human_data/Cyclops_folders/training_output/scROSMAP/cogdx_controls/wAD/ExcitatoryNeurons/TMMs_w_batch/Exc3and5_FiltByEdgeRDefault_fixedipBulkChenZhang_condAndBatchCovs_04ContrVar4EGdefault_noTransferFit_Feb5Redo//downstream_output/PSEA_files/PSEA_AD_cyclers_AR25BHQ1_c2cpkeggv20231Hssymbols
Min geneset size: 5
Max permutations: 10000
Using output img format: pdf
Successfully loaded 197 items
Successfully loaded 186 items
Absolute Path to gene-acrophase file: /Users/henryhollis/Box Sync/Henry_stuff/AD_project/human_data/Cyclops_folders/training_output/scROSMAP/cogdx_controls/wAD/ExcitatoryNeurons/TMMs_w_batch/Exc3and5_FiltByEdgeRDefault_fixedipBulkChenZhang_condAndBatchCovs_04ContrVar4EGdefault_noTransferFit_Feb5Redo//downstream_output/PSEA_files/PSEA_CTL_cyclers_AR25BHQ1.txt
Absolute Path to gmt file: /Users/henryhollis/Box Sync/Henry_stuff/AD_project/scROSMAP/Rscripts/automatic_downstream_analysis/MsigDB_gmts_for_GSEA/c2.cp.kegg.v2023.1.Hs.symbols.gmt
Absolute Path to output folder: /Users/henryhollis/Box Sync/Henry_stuff/AD_project/human_data/Cyclops_folders/training_output/scROSMAP/cogdx_controls/wAD/ExcitatoryNeurons/TMMs_w_batch/Exc3and5_FiltByEdgeRDefault_fixedipBulkChenZhang_condAndBatchCovs_04ContrVar4EGdefault_noTransferFit_Feb5Redo//downstream_output/PSEA_files/PSEA_CTL_cyclers_AR25BHQ1_c2cpkeggv20231Hssymbols
Min geneset size: 5
Max permutations: 10000
Using output img format: pdf
Successfully loaded 204 items
Successfully loaded 186 items
Absolute Path to gene-acrophase file: /Users/henryhollis/Box Sync/Henry_stuff/AD_project/human_data/Cyclops_folders/training_output/scROSMAP/cogdx_controls/wAD/ExcitatoryNeurons/TMMs_w_batch/Exc3and5_FiltByEdgeRDefault_fixedipBulkChenZhang_condAndBatchCovs_04ContrVar4EGdefault_noTransferFit_Feb5Redo//downstream_output/PSEA_files/PSEA_DR_AR25BHQ2_acrodiffs.txt
Absolute Path to gmt file: /Users/henryhollis/Box Sync/Henry_stuff/AD_project/scROSMAP/Rscripts/automatic_downstream_analysis/MsigDB_gmts_for_GSEA/c2.cp.kegg.v2023.1.Hs.symbols.gmt
Absolute Path to output folder: /Users/henryhollis/Box Sync/Henry_stuff/AD_project/human_data/Cyclops_folders/training_output/scROSMAP/cogdx_controls/wAD/ExcitatoryNeurons/TMMs_w_batch/Exc3and5_FiltByEdgeRDefault_fixedipBulkChenZhang_condAndBatchCovs_04ContrVar4EGdefault_noTransferFit_Feb5Redo//downstream_output/PSEA_files/PSEA_DR_AR25BHQ2_acrodiffs_c2cpkeggv20231Hssymbols
Min geneset size: 5
Max permutations: 10000
Using output img format: pdf
Successfully loaded 7 items
Successfully loaded 186 items
Absolute Path to gene-acrophase file: /Users/henryhollis/Box Sync/Henry_stuff/AD_project/human_data/Cyclops_folders/training_output/scROSMAP/cogdx_controls/wAD/ExcitatoryNeurons/TMMs_w_batch/Exc3and5_FiltByEdgeRDefault_fixedipBulkChenZhang_condAndBatchCovs_04ContrVar4EGdefault_noTransferFit_Feb5Redo//downstream_output/PSEA_files/PSEA_AD_cyclers_AR25BHQ1.txt
Absolute Path to gmt file: /Users/henryhollis/Box Sync/Henry_stuff/AD_project/scROSMAP/Rscripts/automatic_downstream_analysis/MsigDB_gmts_for_GSEA/h.all.v2023.1.Hs.symbols.gmt
Absolute Path to output folder: /Users/henryhollis/Box Sync/Henry_stuff/AD_project/human_data/Cyclops_folders/training_output/scROSMAP/cogdx_controls/wAD/ExcitatoryNeurons/TMMs_w_batch/Exc3and5_FiltByEdgeRDefault_fixedipBulkChenZhang_condAndBatchCovs_04ContrVar4EGdefault_noTransferFit_Feb5Redo//downstream_output/PSEA_files/PSEA_AD_cyclers_AR25BHQ1_hallv20231Hssymbols
Min geneset size: 5
Max permutations: 10000
Using output img format: pdf
Successfully loaded 197 items
Successfully loaded 50 items
Absolute Path to gene-acrophase file: /Users/henryhollis/Box Sync/Henry_stuff/AD_project/human_data/Cyclops_folders/training_output/scROSMAP/cogdx_controls/wAD/ExcitatoryNeurons/TMMs_w_batch/Exc3and5_FiltByEdgeRDefault_fixedipBulkChenZhang_condAndBatchCovs_04ContrVar4EGdefault_noTransferFit_Feb5Redo//downstream_output/PSEA_files/PSEA_CTL_cyclers_AR25BHQ1.txt
Absolute Path to gmt file: /Users/henryhollis/Box Sync/Henry_stuff/AD_project/scROSMAP/Rscripts/automatic_downstream_analysis/MsigDB_gmts_for_GSEA/h.all.v2023.1.Hs.symbols.gmt
Absolute Path to output folder: /Users/henryhollis/Box Sync/Henry_stuff/AD_project/human_data/Cyclops_folders/training_output/scROSMAP/cogdx_controls/wAD/ExcitatoryNeurons/TMMs_w_batch/Exc3and5_FiltByEdgeRDefault_fixedipBulkChenZhang_condAndBatchCovs_04ContrVar4EGdefault_noTransferFit_Feb5Redo//downstream_output/PSEA_files/PSEA_CTL_cyclers_AR25BHQ1_hallv20231Hssymbols
Min geneset size: 5
Max permutations: 10000
Using output img format: pdf
Successfully loaded 204 items
Successfully loaded 50 items
Absolute Path to gene-acrophase file: /Users/henryhollis/Box Sync/Henry_stuff/AD_project/human_data/Cyclops_folders/training_output/scROSMAP/cogdx_controls/wAD/ExcitatoryNeurons/TMMs_w_batch/Exc3and5_FiltByEdgeRDefault_fixedipBulkChenZhang_condAndBatchCovs_04ContrVar4EGdefault_noTransferFit_Feb5Redo//downstream_output/PSEA_files/PSEA_DR_AR25BHQ2_acrodiffs.txt
Absolute Path to gmt file: /Users/henryhollis/Box Sync/Henry_stuff/AD_project/scROSMAP/Rscripts/automatic_downstream_analysis/MsigDB_gmts_for_GSEA/h.all.v2023.1.Hs.symbols.gmt
Absolute Path to output folder: /Users/henryhollis/Box Sync/Henry_stuff/AD_project/human_data/Cyclops_folders/training_output/scROSMAP/cogdx_controls/wAD/ExcitatoryNeurons/TMMs_w_batch/Exc3and5_FiltByEdgeRDefault_fixedipBulkChenZhang_condAndBatchCovs_04ContrVar4EGdefault_noTransferFit_Feb5Redo//downstream_output/PSEA_files/PSEA_DR_AR25BHQ2_acrodiffs_hallv20231Hssymbols
Min geneset size: 5
Max permutations: 10000
Using output img format: pdf
Successfully loaded 7 items
Successfully loaded 50 items
[1] 0 0 0 0 0 0

Transcription Factor Analysis

Now I search for transcription factors associated with my various gene lists, i.e, TF’s associated with genes that lose amplitude in AD, etc.

path = getwd()

#create pscan dir if does not exist:
if(!(dir.exists(paste0(path_to_cyclops_ordering, "downstream_output/pscan")))){
  dir.create(paste0(path_to_cyclops_ordering, "downstream_output/pscan") )
}
setwd(paste0(path_to_cyclops_ordering, "downstream_output"))

Warning: The working directory was changed to /Users/henryhollis/Box Sync/Henry_stuff/AD_project/human_data/Cyclops_folders/training_output/scROSMAP/cogdx_controls/wAD/ExcitatoryNeurons/TMMs_w_batch/Exc3and5_FiltByEdgeRDefault_fixedipBulkChenZhang_condAndBatchCovs_04ContrVar4EGdefault_noTransferFit_Feb5Redo/downstream_output inside a notebook chunk. The working directory will be reset when the chunk is finished running. Use the knitr root.dir option in the setup chunk to change the working directory for notebook chunks.

#grab gene lists from enrichR files directory
gene_lists = list.files("./enrichR_files/", pattern = "^(CTL_|AD_|DR_)")

#write pscan input
lapply(gene_lists, write_pscan_input)

Loading required package: AnnotationDbi
Warning: package ‘AnnotationDbi’ was built under R version 4.3.1Loading required package: stats4
Loading required package: BiocGenerics

Attaching package: ‘BiocGenerics’

The following object is masked from ‘package:gridExtra’:

    combine

The following objects are masked from ‘package:lubridate’:

    intersect, setdiff, union

The following objects are masked from ‘package:dplyr’:

    combine, intersect, setdiff, union

The following objects are masked from ‘package:stats’:

    IQR, mad, sd, var, xtabs

The following objects are masked from ‘package:base’:

    anyDuplicated, aperm, append, as.data.frame, basename, cbind, colnames, dirname, do.call, duplicated, eval, evalq, Filter,
    Find, get, grep, grepl, intersect, is.unsorted, lapply, Map, mapply, match, mget, order, paste, pmax, pmax.int, pmin,
    pmin.int, Position, rank, rbind, Reduce, rownames, sapply, setdiff, sort, table, tapply, union, unique, unsplit, which.max,
    which.min

Loading required package: Biobase
Welcome to Bioconductor

    Vignettes contain introductory material; view with 'browseVignettes()'. To cite Bioconductor, see 'citation("Biobase")', and
    for packages 'citation("pkgname")'.

Loading required package: IRanges
Warning: package ‘IRanges’ was built under R version 4.3.1Loading required package: S4Vectors

Attaching package: ‘S4Vectors’

The following objects are masked from ‘package:data.table’:

    first, second

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    second, second<-

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    first, rename

The following object is masked from ‘package:tidyr’:

    expand

The following object is masked from ‘package:utils’:

    findMatches

The following objects are masked from ‘package:base’:

    expand.grid, I, unname


Attaching package: ‘IRanges’

The following object is masked from ‘package:data.table’:

    shift

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    %within%

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    collapse, desc, slice

The following object is masked from ‘package:purrr’:

    reduce


Attaching package: ‘AnnotationDbi’

The following object is masked from ‘package:dplyr’:

    select


'select()' returned 1:many mapping between keys and columns
'select()' returned 1:many mapping between keys and columns
'select()' returned 1:many mapping between keys and columns
'select()' returned 1:many mapping between keys and columns
'select()' returned 1:many mapping between keys and columns

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path_no_space = gsub(" ", "\\ ", path, fixed = TRUE)

setwd("pscan/")
pscan_files = list.files("./pscan_files/", pattern = ".csv$")

sapply(pscan_files, function(x){
  system(paste0("python3 ", path_no_space, "/pscan_requests.py --file pscan_files/" ,x))})

Finished Pscan on AD_CTL_mthd2_cyclers_AR25BHQ1_pscan.csv
Number of genes: 566
Finished Pscan on AD_cyclers_AR25BHQ1_pscan.csv
Number of genes: 340
Finished Pscan on AD_cyclers_AR33BHQ1_pscan.csv
Number of genes: 137
Finished Pscan on CTL_cyclers_AR25BHQ1_pscan.csv
Number of genes: 382
Finished Pscan on CTL_cyclers_AR33BHQ1_pscan.csv
Number of genes: 150
AD_CTL_mthd2_cyclers_AR25BHQ1_pscan.csv           AD_cyclers_AR25BHQ1_pscan.csv           AD_cyclers_AR33BHQ1_pscan.csv 
                                      0                                       0                                       0 
         CTL_cyclers_AR25BHQ1_pscan.csv          CTL_cyclers_AR33BHQ1_pscan.csv 
                                      0                                       0 

isCyclingBHQCutoff_str = str_extract(as.character(isCyclingBHQCutoff), "(?<=\\.)\\d+")

if(exists("deseq_de_filename")){
  setwd("pscan_results")
  pscan_result_files = list.files(pattern = ".csv$")
  print("Searching cycling and DR results for pscan TF")
  sapply(pscan_result_files, augment_tf_file, deseq_de_filename, isCyclingBHQCutoff_str)
  setwd(paste0(path_to_cyclops_ordering, "downstream_output/enrichR_results"))
  directories = list.files()
  enrichR_TF_paths <- lapply(directories, function(dir) list.files(dir, full.names = TRUE, recursive = TRUE, pattern = ".*TRANSFACandJASPARPWMs_BCKGRND.*\\.csv|.*TranscriptionFactorPPIs_BCKGRND.*\\.csv"))
  enrichR_TF_paths = flatten(enrichR_TF_paths) %>% paste0("./", .)  
  sapply(enrichR_TF_paths, augment_tf_file, deseq_de_filename,     isCyclingBHQCutoff_str)
  
}

[1] "Searching cycling and DR results for pscan TF"
                                                                                                                                                                                                                                                                                                                                                                             ./EnrichR_ADCTLmthd2cyclersAR25BHQ1_BCKGRND-EnrichRbackground/ADCTLmthd2cyclersAR25BHQ1_LIBRY-TranscriptionFactorPPIs_BCKGRND-EnrichRbackground.csv 
                                                                  "/Users/henryhollis/Box Sync/Henry_stuff/AD_project/human_data/Cyclops_folders/training_output/scROSMAP/cogdx_controls/wAD/ExcitatoryNeurons/TMMs_w_batch/Exc3and5_FiltByEdgeRDefault_fixedipBulkChenZhang_condAndBatchCovs_04ContrVar4EGdefault_noTransferFit_Feb5Redo/downstream_output/enrichR_results/EnrichR_ADCTLmthd2cyclersAR25BHQ1_BCKGRND-EnrichRbackground/ADCTLmthd2cyclersAR25BHQ1_LIBRY-TranscriptionFactorPPIs_BCKGRND-EnrichRbackground_plots" 
                                                                                                                                                                                                                                                                                                                                                                               ./EnrichR_ADCTLmthd2cyclersAR25BHQ1_BCKGRND-EnrichRbackground/ADCTLmthd2cyclersAR25BHQ1_LIBRY-TRANSFACandJASPARPWMs_BCKGRND-EnrichRbackground.csv 
                                                                    "/Users/henryhollis/Box Sync/Henry_stuff/AD_project/human_data/Cyclops_folders/training_output/scROSMAP/cogdx_controls/wAD/ExcitatoryNeurons/TMMs_w_batch/Exc3and5_FiltByEdgeRDefault_fixedipBulkChenZhang_condAndBatchCovs_04ContrVar4EGdefault_noTransferFit_Feb5Redo/downstream_output/enrichR_results/EnrichR_ADCTLmthd2cyclersAR25BHQ1_BCKGRND-EnrichRbackground/ADCTLmthd2cyclersAR25BHQ1_LIBRY-TRANSFACandJASPARPWMs_BCKGRND-EnrichRbackground_plots" 
                                                                                                                                                                                                                                                                                                                                                                                             ./EnrichR_ADcyclersAR25BHQ1_BCKGRND-EnrichRbackground/ADcyclersAR25BHQ1_LIBRY-TranscriptionFactorPPIs_BCKGRND-EnrichRbackground.csv 
                                                                                  "/Users/henryhollis/Box Sync/Henry_stuff/AD_project/human_data/Cyclops_folders/training_output/scROSMAP/cogdx_controls/wAD/ExcitatoryNeurons/TMMs_w_batch/Exc3and5_FiltByEdgeRDefault_fixedipBulkChenZhang_condAndBatchCovs_04ContrVar4EGdefault_noTransferFit_Feb5Redo/downstream_output/enrichR_results/EnrichR_ADcyclersAR25BHQ1_BCKGRND-EnrichRbackground/ADcyclersAR25BHQ1_LIBRY-TranscriptionFactorPPIs_BCKGRND-EnrichRbackground_plots" 
                                                                                                                                                                                                                                                                                                                                                                                               ./EnrichR_ADcyclersAR25BHQ1_BCKGRND-EnrichRbackground/ADcyclersAR25BHQ1_LIBRY-TRANSFACandJASPARPWMs_BCKGRND-EnrichRbackground.csv 
                                                                                    "/Users/henryhollis/Box Sync/Henry_stuff/AD_project/human_data/Cyclops_folders/training_output/scROSMAP/cogdx_controls/wAD/ExcitatoryNeurons/TMMs_w_batch/Exc3and5_FiltByEdgeRDefault_fixedipBulkChenZhang_condAndBatchCovs_04ContrVar4EGdefault_noTransferFit_Feb5Redo/downstream_output/enrichR_results/EnrichR_ADcyclersAR25BHQ1_BCKGRND-EnrichRbackground/ADcyclersAR25BHQ1_LIBRY-TRANSFACandJASPARPWMs_BCKGRND-EnrichRbackground_plots" 
                                                                                                                                                                                                                                                                                                                                                                                             ./EnrichR_ADcyclersAR33BHQ1_BCKGRND-EnrichRbackground/ADcyclersAR33BHQ1_LIBRY-TranscriptionFactorPPIs_BCKGRND-EnrichRbackground.csv 
                                                                                  "/Users/henryhollis/Box Sync/Henry_stuff/AD_project/human_data/Cyclops_folders/training_output/scROSMAP/cogdx_controls/wAD/ExcitatoryNeurons/TMMs_w_batch/Exc3and5_FiltByEdgeRDefault_fixedipBulkChenZhang_condAndBatchCovs_04ContrVar4EGdefault_noTransferFit_Feb5Redo/downstream_output/enrichR_results/EnrichR_ADcyclersAR33BHQ1_BCKGRND-EnrichRbackground/ADcyclersAR33BHQ1_LIBRY-TranscriptionFactorPPIs_BCKGRND-EnrichRbackground_plots" 
                                                                                                                                                                                                                                                                                                                                                                                               ./EnrichR_ADcyclersAR33BHQ1_BCKGRND-EnrichRbackground/ADcyclersAR33BHQ1_LIBRY-TRANSFACandJASPARPWMs_BCKGRND-EnrichRbackground.csv 
                                                                                    "/Users/henryhollis/Box Sync/Henry_stuff/AD_project/human_data/Cyclops_folders/training_output/scROSMAP/cogdx_controls/wAD/ExcitatoryNeurons/TMMs_w_batch/Exc3and5_FiltByEdgeRDefault_fixedipBulkChenZhang_condAndBatchCovs_04ContrVar4EGdefault_noTransferFit_Feb5Redo/downstream_output/enrichR_results/EnrichR_ADcyclersAR33BHQ1_BCKGRND-EnrichRbackground/ADcyclersAR33BHQ1_LIBRY-TRANSFACandJASPARPWMs_BCKGRND-EnrichRbackground_plots" 
                                                                                                                                                                                                                                                                                                                                                                                           ./EnrichR_CTLcyclersAR25BHQ1_BCKGRND-EnrichRbackground/CTLcyclersAR25BHQ1_LIBRY-TranscriptionFactorPPIs_BCKGRND-EnrichRbackground.csv 
                                                                                "/Users/henryhollis/Box Sync/Henry_stuff/AD_project/human_data/Cyclops_folders/training_output/scROSMAP/cogdx_controls/wAD/ExcitatoryNeurons/TMMs_w_batch/Exc3and5_FiltByEdgeRDefault_fixedipBulkChenZhang_condAndBatchCovs_04ContrVar4EGdefault_noTransferFit_Feb5Redo/downstream_output/enrichR_results/EnrichR_CTLcyclersAR25BHQ1_BCKGRND-EnrichRbackground/CTLcyclersAR25BHQ1_LIBRY-TranscriptionFactorPPIs_BCKGRND-EnrichRbackground_plots" 
                                                                                                                                                                                                                                                                                                                                                                                             ./EnrichR_CTLcyclersAR25BHQ1_BCKGRND-EnrichRbackground/CTLcyclersAR25BHQ1_LIBRY-TRANSFACandJASPARPWMs_BCKGRND-EnrichRbackground.csv 
                                                                                  "/Users/henryhollis/Box Sync/Henry_stuff/AD_project/human_data/Cyclops_folders/training_output/scROSMAP/cogdx_controls/wAD/ExcitatoryNeurons/TMMs_w_batch/Exc3and5_FiltByEdgeRDefault_fixedipBulkChenZhang_condAndBatchCovs_04ContrVar4EGdefault_noTransferFit_Feb5Redo/downstream_output/enrichR_results/EnrichR_CTLcyclersAR25BHQ1_BCKGRND-EnrichRbackground/CTLcyclersAR25BHQ1_LIBRY-TRANSFACandJASPARPWMs_BCKGRND-EnrichRbackground_plots" 
                                                                                                                                                                                                                                                                                                                                                                                           ./EnrichR_CTLcyclersAR33BHQ1_BCKGRND-EnrichRbackground/CTLcyclersAR33BHQ1_LIBRY-TranscriptionFactorPPIs_BCKGRND-EnrichRbackground.csv 
                                                                                "/Users/henryhollis/Box Sync/Henry_stuff/AD_project/human_data/Cyclops_folders/training_output/scROSMAP/cogdx_controls/wAD/ExcitatoryNeurons/TMMs_w_batch/Exc3and5_FiltByEdgeRDefault_fixedipBulkChenZhang_condAndBatchCovs_04ContrVar4EGdefault_noTransferFit_Feb5Redo/downstream_output/enrichR_results/EnrichR_CTLcyclersAR33BHQ1_BCKGRND-EnrichRbackground/CTLcyclersAR33BHQ1_LIBRY-TranscriptionFactorPPIs_BCKGRND-EnrichRbackground_plots" 
                                                                                                                                                                                                                                                                                                                                                                                             ./EnrichR_CTLcyclersAR33BHQ1_BCKGRND-EnrichRbackground/CTLcyclersAR33BHQ1_LIBRY-TRANSFACandJASPARPWMs_BCKGRND-EnrichRbackground.csv 
                                                                                  "/Users/henryhollis/Box Sync/Henry_stuff/AD_project/human_data/Cyclops_folders/training_output/scROSMAP/cogdx_controls/wAD/ExcitatoryNeurons/TMMs_w_batch/Exc3and5_FiltByEdgeRDefault_fixedipBulkChenZhang_condAndBatchCovs_04ContrVar4EGdefault_noTransferFit_Feb5Redo/downstream_output/enrichR_results/EnrichR_CTLcyclersAR33BHQ1_BCKGRND-EnrichRbackground/CTLcyclersAR33BHQ1_LIBRY-TRANSFACandJASPARPWMs_BCKGRND-EnrichRbackground_plots" 
                                                                                                                                                                                                                                                                                                                                                                                   ./EnrichR_diffmesorallgenesBHQ05_BCKGRND-EnrichRbackground/diffmesorallgenesBHQ05_LIBRY-TranscriptionFactorPPIs_BCKGRND-EnrichRbackground.csv 
                                                                        "/Users/henryhollis/Box Sync/Henry_stuff/AD_project/human_data/Cyclops_folders/training_output/scROSMAP/cogdx_controls/wAD/ExcitatoryNeurons/TMMs_w_batch/Exc3and5_FiltByEdgeRDefault_fixedipBulkChenZhang_condAndBatchCovs_04ContrVar4EGdefault_noTransferFit_Feb5Redo/downstream_output/enrichR_results/EnrichR_diffmesorallgenesBHQ05_BCKGRND-EnrichRbackground/diffmesorallgenesBHQ05_LIBRY-TranscriptionFactorPPIs_BCKGRND-EnrichRbackground_plots" 
                                                                                                                                                                                                                                                                                                                                                                                     ./EnrichR_diffmesorallgenesBHQ05_BCKGRND-EnrichRbackground/diffmesorallgenesBHQ05_LIBRY-TRANSFACandJASPARPWMs_BCKGRND-EnrichRbackground.csv 
                                                                          "/Users/henryhollis/Box Sync/Henry_stuff/AD_project/human_data/Cyclops_folders/training_output/scROSMAP/cogdx_controls/wAD/ExcitatoryNeurons/TMMs_w_batch/Exc3and5_FiltByEdgeRDefault_fixedipBulkChenZhang_condAndBatchCovs_04ContrVar4EGdefault_noTransferFit_Feb5Redo/downstream_output/enrichR_results/EnrichR_diffmesorallgenesBHQ05_BCKGRND-EnrichRbackground/diffmesorallgenesBHQ05_LIBRY-TRANSFACandJASPARPWMs_BCKGRND-EnrichRbackground_plots" 
                                                                                                                                                                                                                                                                                                                                       ./EnrichR_DRcyclersCyclingBHQ1AR25DRBHQ2_BCKGRND-diffrhythmsCyclingBHQ1AmpRatio25/DRcyclersCyclingBHQ1AR25DRBHQ2_LIBRY-TRANSFACandJASPARPWMs_BCKGRND-diffrhythmsCyclingBHQ1AmpRatio25.csv 
                            "/Users/henryhollis/Box Sync/Henry_stuff/AD_project/human_data/Cyclops_folders/training_output/scROSMAP/cogdx_controls/wAD/ExcitatoryNeurons/TMMs_w_batch/Exc3and5_FiltByEdgeRDefault_fixedipBulkChenZhang_condAndBatchCovs_04ContrVar4EGdefault_noTransferFit_Feb5Redo/downstream_output/enrichR_results/EnrichR_DRcyclersCyclingBHQ1AR25DRBHQ2_BCKGRND-diffrhythmsCyclingBHQ1AmpRatio25/DRcyclersCyclingBHQ1AR25DRBHQ2_LIBRY-TRANSFACandJASPARPWMs_BCKGRND-diffrhythmsCyclingBHQ1AmpRatio25_plots" 
                                                                                                                                                                                                                                                                                                               ./EnrichR_DRcyclersmthd2CyclingBHQ1AR25DRBHQ2_BCKGRND-diffrhythmsmethod2CyclingBHQ1AmpRatio25/DRcyclersmthd2CyclingBHQ1AR25DRBHQ2_LIBRY-TRANSFACandJASPARPWMs_BCKGRND-diffrhythmsmethod2CyclingBHQ1AmpRatio25.csv 
    "/Users/henryhollis/Box Sync/Henry_stuff/AD_project/human_data/Cyclops_folders/training_output/scROSMAP/cogdx_controls/wAD/ExcitatoryNeurons/TMMs_w_batch/Exc3and5_FiltByEdgeRDefault_fixedipBulkChenZhang_condAndBatchCovs_04ContrVar4EGdefault_noTransferFit_Feb5Redo/downstream_output/enrichR_results/EnrichR_DRcyclersmthd2CyclingBHQ1AR25DRBHQ2_BCKGRND-diffrhythmsmethod2CyclingBHQ1AmpRatio25/DRcyclersmthd2CyclingBHQ1AR25DRBHQ2_LIBRY-TRANSFACandJASPARPWMs_BCKGRND-diffrhythmsmethod2CyclingBHQ1AmpRatio25_plots" 
                                                                                                                                                                                                                                                                                                                                   ./EnrichR_DRgainAmpADCyclingBHQ1AR25DRBHQ2_BCKGRND-diffrhythmsCyclingBHQ1AmpRatio25/DRgainAmpADCyclingBHQ1AR25DRBHQ2_LIBRY-TRANSFACandJASPARPWMs_BCKGRND-diffrhythmsCyclingBHQ1AmpRatio25.csv 
                        "/Users/henryhollis/Box Sync/Henry_stuff/AD_project/human_data/Cyclops_folders/training_output/scROSMAP/cogdx_controls/wAD/ExcitatoryNeurons/TMMs_w_batch/Exc3and5_FiltByEdgeRDefault_fixedipBulkChenZhang_condAndBatchCovs_04ContrVar4EGdefault_noTransferFit_Feb5Redo/downstream_output/enrichR_results/EnrichR_DRgainAmpADCyclingBHQ1AR25DRBHQ2_BCKGRND-diffrhythmsCyclingBHQ1AmpRatio25/DRgainAmpADCyclingBHQ1AR25DRBHQ2_LIBRY-TRANSFACandJASPARPWMs_BCKGRND-diffrhythmsCyclingBHQ1AmpRatio25_plots" 
                                                                                                                                                                                                                                                                                                           ./EnrichR_DRmthd2gainAmpADCyclingBHQ1AR25DRBHQ2_BCKGRND-diffrhythmsmethod2CyclingBHQ1AmpRatio25/DRmthd2gainAmpADCyclingBHQ1AR25DRBHQ2_LIBRY-TRANSFACandJASPARPWMs_BCKGRND-diffrhythmsmethod2CyclingBHQ1AmpRatio25.csv 
"/Users/henryhollis/Box Sync/Henry_stuff/AD_project/human_data/Cyclops_folders/training_output/scROSMAP/cogdx_controls/wAD/ExcitatoryNeurons/TMMs_w_batch/Exc3and5_FiltByEdgeRDefault_fixedipBulkChenZhang_condAndBatchCovs_04ContrVar4EGdefault_noTransferFit_Feb5Redo/downstream_output/enrichR_results/EnrichR_DRmthd2gainAmpADCyclingBHQ1AR25DRBHQ2_BCKGRND-diffrhythmsmethod2CyclingBHQ1AmpRatio25/DRmthd2gainAmpADCyclingBHQ1AR25DRBHQ2_LIBRY-TRANSFACandJASPARPWMs_BCKGRND-diffrhythmsmethod2CyclingBHQ1AmpRatio25_plots" 

Write KEGG Files

Write Files for Kegg Pathway Diagrams

entrez_dict = readxl::read_excel("scROSMAP_ENSEMBL_ENTREZ_dict.xlsx")

setwd(path_to_cyclops_ordering)

Warning: The working directory was changed to /Users/henryhollis/Box Sync/Henry_stuff/AD_project/human_data/Cyclops_folders/training_output/scROSMAP/cogdx_controls/wAD/ExcitatoryNeurons/TMMs_w_batch/Exc3and5_FiltByEdgeRDefault_fixedipBulkChenZhang_condAndBatchCovs_04ContrVar4EGdefault_noTransferFit_Feb5Redo inside a notebook chunk. The working directory will be reset when the chunk is finished running. Use the knitr root.dir option in the setup chunk to change the working directory for notebook chunks.

isCyclingBHQCutoff_str = str_extract(as.character(isCyclingBHQCutoff), "(?<=\\.)\\d+")

if (!(dir.exists("downstream_output/KEGG_map_diagrams"))){
  dir.create("downstream_output/KEGG_map_diagrams")
}
write_kegg_map_files(paste0("downstream_output/diff_rhythms_CyclingBHQ", isCyclingBHQCutoff_str, "AmpRatio25.csv"),"Log_AD_CTL_ampRatio", trans_dict = entrez_dict,BHQ_cutoff = 0.2)

Metabolon

if (!(dir.exists(paste0(path_to_cyclops_ordering, "/downstream_output/metabolon")))){
  dir.create(paste0(path_to_cyclops_ordering, "/downstream_output/metabolon"))
}

order_metabolon(metabolon_filename, metabolon_datakey, path_to_rosmap_clin, path_to_cyclops_ordering)

Running is_cycling() on cond_subset: cond_0
Running is_cycling() on cond_subset: cond_1
Running is_cycling_method2() (analogue of compareRhythms) on all subjects
Running diff_rhyth() on seedlist of size: 263
Running diff_rhyth() on seedlist of size: 292[[1]]
[1] "plots/100002284_gene_trace.png"

[[2]]
[1] "plots/100000265_gene_trace.png"

[[3]]
[1] "plots/100002458_gene_trace.png"

[[4]]
[1] "plots/799_gene_trace.png"

[[5]]
[1] "plots/100000265_gene_trace.png"

[[6]]
[1] "plots/460_gene_trace.png"

Warning: The working directory was changed to /Users/henryhollis/Box Sync/Henry_stuff/AD_project/human_data/Cyclops_folders/training_output/scROSMAP/cogdx_controls/wAD/ExcitatoryNeurons/TMMs_w_batch/Exc3and5_FiltByEdgeRDefault_fixedipBulkChenZhang_condAndBatchCovs_04ContrVar4EGdefault_noTransferFit_Feb5Redo/downstream_output/metabolon inside a notebook chunk. The working directory will be reset when the chunk is finished running. Use the knitr root.dir option in the setup chunk to change the working directory for notebook chunks.

Proteomics

Make sure you have downloaded syn32835854, syn21266449, and syn51389605. You can download programmatically with python or R synapse client. Make sure you specify the path to those downloads with proteiomics_file_path.

proteiomics_file_path = "~/Box Sync/Henry_stuff/AD_project/scROSMAP/Proteomics/SynapseDownloads/"
tmt_filename = "2b.unlogged_relative_abundance-ROSMAP610.R1+R2-2xTAMPORcorrected.csv" #syn51389605
# tmt_filename = "3.Regressed(age.sex.PMI-noDxProtect)_cleanDat-7814x610-ROSMAP610_R1+R2(Uniprot2019multiconsensus).csv"

#make sure you have downloaded syn32835854,  syn21266449, and syn51389605
if (!(dir.exists(paste0(path_to_cyclops_ordering, "/downstream_output/proteomics")))){
  dir.create(paste0(path_to_cyclops_ordering, "/downstream_output/proteomics"))
}

order_prot(proteiomics_file_path, tmt_filename, path_to_rosmap_clin, path_to_cyclops_ordering)

New names:New names:

Running is_cycling() on cond_subset: cond_0

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Running is_cycling() on cond_subset: cond_1

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Running is_cycling_method2() (analogue of compareRhythms) on all subjects

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Running Mesor_differences()

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Running diff_rhyth() on seedlist of size: 753
Running diff_rhyth() on seedlist of size: 942[[1]]
[1] "plots/KIF5A|Q12840_gene_trace.png"

[[2]]
[1] "plots/RPS6KA5|O75582_gene_trace.png"

[[3]]
[1] "plots/FKBP5|Q13451_gene_trace.png"

[[4]]
[1] "plots/BOLA2; BOLA2B|Q9H3K6_gene_trace.png"

[[5]]
[1] "plots/OXSR1|O95747_gene_trace.png"

[[6]]
[1] "plots/PEX11B|O96011_gene_trace.png"

Warning: The working directory was changed to /Users/henryhollis/Box Sync/Henry_stuff/AD_project/human_data/Cyclops_folders/training_output/scROSMAP/cogdx_controls/wAD/ExcitatoryNeurons/TMMs_w_batch/Exc3and5_FiltByEdgeRDefault_fixedipBulkChenZhang_condAndBatchCovs_04ContrVar4EGdefault_noTransferFit_Feb5Redo/downstream_output/proteomics inside a notebook chunk. The working directory will be reset when the chunk is finished running. Use the knitr root.dir option in the setup chunk to change the working directory for notebook chunks.

Plot Other Proteins

plot_list = c("BPNT1|O95861", "PPM1B|O75688", "BLVRA|P53004", "HDHD2|Q9H0R4")

proteiomics_file_path = "~/Box Sync/Henry_stuff/AD_project/scROSMAP/Proteomics/SynapseDownloads/"
tmt_filename = "2b.unlogged_relative_abundance-ROSMAP610.R1+R2-2xTAMPORcorrected.csv" #syn51389605
# tmt_filename = "3.Regressed(age.sex.PMI-noDxProtect)_cleanDat-7814x610-ROSMAP610_R1+R2(Uniprot2019multiconsensus).csv"

plot_prot(proteiomics_file_path, tmt_filename, path_to_rosmap_clin, path_to_cyclops_ordering, c("RPL10|P27635", "RPL32|P62910", "NDUFB1|O75438"), percentile = percentile)

fGSEA on proteomics

path = getwd()
setwd(path_to_cyclops_ordering)

Warning: The working directory was changed to /Users/henryhollis/Box Sync/Henry_stuff/AD_project/human_data/Cyclops_folders/training_output/scROSMAP/cogdx_controls/wAD/ExcitatoryNeurons/TMMs_w_batch/Exc3and5_FiltByEdgeRDefault_fixedipBulkChenZhang_condAndBatchCovs_04ContrVar4EGdefault_noTransferFit_Feb5Redo inside a notebook chunk. The working directory will be reset when the chunk is finished running. Use the knitr root.dir option in the setup chunk to change the working directory for notebook chunks.

if (!(dir.exists("downstream_output/proteomics/fGSEA"))){
  dir.create("downstream_output/proteomics/fGSEA")
  dir.create("downstream_output/proteomics/fGSEA/rnk_files")
  dir.create("downstream_output/proteomics/fGSEA/fGSEA_results")
}

isCyclingBHQCutoff_str = str_extract(as.character(isCyclingBHQCutoff), "(?<=\\.)\\d+")

#path of parent folder
setwd(path)

write_prot_rnks(path_to_cyclops_ordering)

[1] "Creating rnk files for fGSEA."

#Pathways, downloaded from MsigDB
pathways <- c(gmtPathways("./MsigDB_gmts_for_GSEA/c2.cp.kegg.v2023.1.Hs.symbols.gmt"), gmtPathways("./MsigDB_gmts_for_GSEA/h.all.v2023.1.Hs.symbols.gmt"))


gene_remapping_dict = read.delim("./MsigDB_gmts_for_GSEA/Human_UniProt_IDs_MSigDB.v2023.2.Hs.chip", sep = '\t')

setwd(paste0(path_to_cyclops_ordering,"/downstream_output/proteomics/fGSEA/rnk_files"))

rnk_files = list.files(pattern = ".rnk")

run_fgsea(rnk_files, gene_remapping_dict, pathways)

[1] "../fGSEA_results/AD_cyclers_minusLogPRanked.csv"
[1] "Used pos fGSEA score type"

Saving 7 x 7 in image

[1] "../fGSEA_results/CTL_cyclers_minusLogPRanked.csv"
[1] "Used pos fGSEA score type"
[1] "../fGSEA_results/diff_mesor_(AD-CTL)ranked.csv"
[1] "Used std fGSEA score type"
[1] "../fGSEA_results/DR_cyclers_Log(AD-CTL)ranked.csv"
[1] "Used std fGSEA score type"
[1] "../fGSEA_results/DR_cyclers_minusLogPRanked.csv"
[1] "Used pos fGSEA score type"
                           AD_cyclers_minusLogPRanked.rnk                           CTL_cyclers_minusLogPRanked.rnk 
  "../fGSEA_results/plots/AD_cyclers_minusLogPRanked.png"  "../fGSEA_results/plots/CTL_cyclers_minusLogPRanked.png" 
                            diff_mesor_(AD-CTL)ranked.rnk                          DR_cyclers_Log(AD-CTL)ranked.rnk 
   "../fGSEA_results/plots/diff_mesor_(AD-CTL)ranked.png" "../fGSEA_results/plots/DR_cyclers_Log(AD-CTL)ranked.png" 
                           DR_cyclers_minusLogPRanked.rnk 
  "../fGSEA_results/plots/DR_cyclers_minusLogPRanked.png"